Investigating the Role of the RNA Binding Protein LIN28 in The

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November 2018 Investigating the Role of the RNA Binding Protein LIN28 in the Human Placenta: Implications for Preeclampsia John Canfield University of South Florida, [email protected]

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Investigating the Role of the RNA Binding Protein LIN28 in the Human Placenta:

Implications for Preeclampsia

John Canfield

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Pharmacology and Physiology College of Medicine University of South Florida

Major Professor: Hana Totary-Jain, Ph.D. Bruce Lindsey, Ph.D. Jay Dean, Ph.D. Narasaiah Kolliputi, Ph.D

Date of Approval: November 9, 2018

Keywords: trophoblast, hypoxia, invasion, DOHaD

Acknowledgments

I would like to express my thankfulness to my mentor, Dr. Hana Totary-Jain, for her continuous support, mentoring, and insight as I completed my graduate studies. Her dedication to my training ensured my success and greatly helped in the completion of this dissertation. Her door was always open, and no question was too small or too big. This comprehensive and insightful guidance has put me on a great path to becoming a successful scientist and my gratitude to her will last long after I move on from the lab.

I would also like to thank the members of my dissertation committee: Dr. Bruce

Lindsey, Dr. Jay Dean, and Dr. Narasaiah Kolliputi. Their guidance and assistance along this journey have been a tremendous help and has assisted me as I grow into a successful scientist and as a professional in general. The many committee meetings and general conversations were always pleasant and productive, and for that I thank them.

I would also like to thank Dr. Charles Lockwood and his lab for always being available for questions and conversations. Without the skills and knowledge gained by working with Dr. Lockwood and Dr. Umit Kayisli, any many others in the lab, this dissertation would not have been possible. I am very appreciative of their assistance and

I will be always grateful that they took time out of their own research to assist in mine.

Furthermore, I would like to express my thanks to Dr. John Tsibris for always being available to provide tissue samples for analysis, and for providing constructive conversation. The many conversations about science, professionalism, and life in general were always pleasant and enjoyable. In addition, I would like to thank Dr. Ronald Magness and Dr. Fred Schatz for always being available for conversation and for providing excellent feedback on abstract and manuscript submissions. Also, I would like to express thanks to Dr. Ananth Karumanchi for providing RNA samples for analysis for this dissertation.

Thank you to the members of my lab, Ezinne, Jeff, and Jay, for always being available for assistance and for a lot of laughs during our time in the lab. In addition to always being willing to assist on experiments and lab work, these other members of my lab made day-to-day work more enjoyable and pleasant.

Finally, I would like to thank my wife, Clare, for her continued love and support over the years. Without her motivation and assurance, I simply would not be where I am today. Whether it is talking science, troubleshooting experiments, or just talking about life in general, she is always an open ear and always has insightful advice and conversation.

Her intelligence and passion are inspiring, and I am forever grateful to have her by my side through this journey of life.

Table of Contents

List of Tables ...... iv

List of Figures ...... v

List of Abbreviations ...... vii

Abstract ...... xi

Chapter One: Trophoblast Differentiation and Preeclampsia ...... 1 Preeclampsia ...... 1 Risk factors associated with preeclampsia ...... 2 Long- and short-term adverse health effects associated with preeclampsia ...... 3 Decidualization ...... 3 Development of the trophectoderm and inner cell mass ...... 5 The placenta ...... 9 Placentation in early pregnancy ...... 9 Interstitial EVT invasion ...... 10 Vascular EVT invasion ...... 11 Syncytialization ...... 13 Impaired vascular remodeling in preeclampsia...... 15 Soluble FLT-1 in preeclampsia ...... 18 Soluble endoglin in preeclampsia ...... 19 C19MC in the placenta and preeclampsia ...... 20 Inflammation in preeclampsia ...... 20

Chapter Two: The RNA Binding Protein LIN28 ...... 22 Introduction to LIN28 ...... 22 Regulation of Let-7 by LIN28A and LIN28B ...... 25 Gene regulation by LIN28A and LIN28B independent of Let-7 ...... 28 Regulation of cellular growth and metabolism by LIN28A and LIN28B ...... 29 Upstream regulation of LIN28A and LIN28B ...... 30 LIN28A and LIN28B in embryogenesis and pluripotent stem cells ...... 31 LIN28A and LIN28B in cancer and cellular invasion ...... 32 Gap in knowledge and purpose of this dissertation ...... 34

Chapter Three: Materials and Methods ...... 36 Ethical approval ...... 36 Criteria for inclusion in the study ...... 36

i Placental tissue collection ...... 37 Isolation and culturing of primary DCs ...... 37 Isolation and culturing of primary CTs ...... 38 Isolation and culturing of primary STs ...... 38 Immunohistochemistry ...... 39 Quantification of immunohistochemistry ...... 39 Cell culture...... 40 Cell line authentication ...... 40 Transfections ...... 40 Cell proliferation analysis ...... 41 Hypoxia experiments ...... 41 RNA extractions ...... 42 Protein extractions ...... 42 Immunoblotting ...... 43 qRT-PCR ...... 43 Scratch wound assays ...... 44 Trans-well migration assays ...... 44 Statistical analyses ...... 45

Chapter Four: Decreased LIN28B in Preeclampsia Impairs Human Trophoblast Differentiation and Migration ...... 48 Introduction ...... 48 Results ...... 50 LIN28B is the predominant LIN28 paralog expressed in human term placentas ...... 50 LIN28B is expressed significantly higher in CTs and STs compared to DCs in the human term placenta ...... 52 In situ LIN28B protein is decreased in placentas from PE complicated pregnancies ...... 52 LIN28B mRNA and protein expression is reduced in the placentas of PE complicated pregnancies ...... 55 The Let-7/LIN28B ratio is increased in the placenta of PE complicated pregnancies ...... 58 LIN28B is highly expressed in syncytial sprouts and invasive EVTs of first trimester human placentas ...... 58 LIN28B increased trophoblast migration and invasion in vitro ...... 59 Decreased LIN28B expression reduces trophoblast proliferation ...... 64 LIN28B expression regulates trophoblast differentiation ...... 64 LIN28B expression regulates inflammation ...... 65 Decreased LIN28B expression reduces the expression of ELABELA ...... 70 LIN28B regulates the placenta and ESC specific chromosome 19 miRNA cluster ...... 70 Hypoxia decreases LIN28B expression in BEWO and JEG3 cells ...... 71 Hypoxia decreases LIN28A expression in BEWO and JEG3 cells ...... 76 Hypoxia increases Let-7a and Let-7g expression in BEWO and JEG3

ii cells ...... 76

Hypoxia decreases SYN-1 and ELABELA and increases TNFα in JEG3 and BEWO cells ...... 76 Discussion ...... 82

Chapter Five: Limitations and Overall Conclusions ...... 96 Study limitations ...... 96 Overall conclusions ...... 98

References ...... 102

Appendix A ...... 124 JEG3 ATCC cell line authentication STR analysis profile report...... 124 HTR8/SVneo cell line authentication STR analysis profile report ...... 125 BEWO cell line authentication STR analysis profile report ...... 126 University of South Florida IRB approval ...... 127 Copyright permission from the FASEB Journal ...... 129

iii

List of Tables

Table 1: Demographic data and clinical characteristics of the subjects obtained from the University of South Florida for this study ...... 46

Table 2: Demographic data and clinical characteristics of the subjects obtained from the Harvard Medical School for this study ...... 47

List of Figures

Figure 1: Simplified schematic representation of blastocyst prior to implantation ...... 8

Figure 2: Simplified schematic showing spiral artery remodeling by invasive EVTs ...... 17

Figure 3: Schematic representation of LIN28A and LIN28B protein ...... 24

Figure 4: Mechanism of Let-7 repression by LIN28A and LIN28B ...... 27

Figure 5: LIN28B is the predominant LIN28 paralog expressed in human placentas ...... 51

Figure 6: LIN28B is expressed significantly higher in CTs and STs compared to DCs in the human term placenta ...... 53

Figure 7: in situ LIN28B protein is decreased in placentas from PE pregnancies ...... 54

Figure 8: LIN28B protein and mRNA is decreased in the placenta of PE complicated pregnancies ...... 56

Figure 9: LIN28A protein and mRNA expression is unchanged in the placentas of PE complicated pregnancies ...... 57

Figure 10: The Let-7/LIN28B ratio trends towards an increase in the placenta of PE complicated pregnancies ...... 60

Figure 11: LIN28B is highly expressed in syncytial sprouts and invasive trophoblasts in first trimester placentas ...... 61

Figure 12: LIN28B increases HTR8/SVneo cell migration ...... 62

Figure 13: LIN28B increases HTR8/SVneo cell invasion ...... 63

Figure 14: Transient knockdown of LIN28B in JEG3 cells and transient overexpression of LIN28B in HTR8/SVneo cells ...... 66

Figure 15: LIN28B expression regulates trophoblast proliferation ...... 67

v Figure 16: LIN28B expression regulates trophoblast differentiation ...... 68

Figure 17: LIN28B expression regulates TNFα expression ...... 69

Figure 18: Decreased LIN28B expression reduces ELABELA expression ...... 72

Figure 19: Decreased LIN28B results in reduced expression of the C19MC ...... 73

Figure 20: Hypoxia reduces LIN28B protein and mRNA expression in JEG3 cells ...... 74

Figure 21: Hypoxia reduces LIN28B protein and mRNA expression in BEWO cells ...... 75

Figure 22: Hypoxia reduces LIN28A protein and mRNA expression in JEG3 cells ...... 78

Figure 23: Hypoxia reduces LIN28A protein and mRNA expression in BEWO cells ...... 79

Figure 24: Hypoxia increases Let-7a and Let-7g in JEG3 and BEWO cells ...... 80

Figure 25: Hypoxia reduces SYN-1 and ELABELA and increases TNFα in trophoblasts ...... 81

Figure 26: Schematic model of the contribution of LIN28B to the normal physiology of pregnancy and the pathological conditions of PE ...... 95

List of Abbreviations

BMI Body Mass Index

CS Cesarean section

C19MC Chromosome 19 microRNA Cluster cAMP Cyclic Adenosine Monophosphate

CCHC Cysteine Cysteine Histidine Cysteine domain

CDX2 Caudal Type Homeobox 2

CHIP-seq Chromatin Immunoprecipitation Sequencing

CMV Cytomegalovirus

CLIP-seq Cross-linking Immunoprecipitation Sequencing

CSD Cold-shock Domain

CTs Cytotrophoblast Cells

DCs Decidual Cells dNK Decidual Natural Killer Cells

DOHaD Developmental Origin of Health and Disease

EMT Epithelial to Mesenchymal Transition

Eng Endoglin eNOS Endothelial Nitric Oxide Synthase

EnSCs Endometrial Stromal Cells

ESCs Embryonic Stem Cells

EVTs Extravillous Trophoblasts

vii FBS Fetal Bovine Serum

FGF-4 Fibroblast Growth Factor 4

FLT-1 Fms Related Tyrosine Kinase 1

GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase

GFP Green Fluorescent Protein

GWAS Genome-wide Association Study

HELLP Hemolysis, Elevated Liver Enzymes, Low Platelet Count

HIV Human immunodeficiency virus

Hmga1 High Motility Group AT-hook 1

Hmga2 High Motility Group AT-hook 2

HSV-2 Herpes simplex virus-2

ICM Inner Cell Mass

IGF-2 Insulin-like Growth Factor 2

IRB Institutional Review Board

ITGβ4 Integrin Subunit Beta 4

IUGR Intrauterine Growth Restriction miRNA MicroRNA

MMPs Matrix Metalloproteinases

MPA Medroxyprogesterone Acetate mTOR Mammalian Target of Rapamycin

PAR-clip Photoactivatable Ribonucleoside-Enhanced Crosslinking

and Immunoprecipitation

PBS Phosphate-buffered Saline

viii PlGF Placental growth factor

PE Preeclampsia

PECAM-1 Platelet Endothelial Cell Adhesion Molecule 1

PI3K Phosphoinositide 3-Kinase

PKA Protein Kinase A qRT-PCR Quantitative Reverse Transcription Polymerase Chain

Reaction

RRID Research Resource Identifiers

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

SEM Standard Error of the Mean sEng Soluble Endoglin sFLT-1 Soluble Fms Related Tyrosine Kinase 1

SILAC Stable isotope labeling by amino acids in cell culture

STR Short Tandem Repeat

STs Syncytiotrophoblast Cells

SYN-1 Syncytin 1

SYN-2 Syncytin 2

TE Trophectoderm

Tead4 TEA Domain Transcription Factor 4

TGFβ1 Transforming Growth Factor Beta 1

TGFβ3 Transforming Growth Factor Beta 2

TNFα Tumor Necrosis Factor Alpha

TUT4 Terminal Uridylyltransferase 4

ix VECAM-1 Vascular Cell Adhesion Molecule 1

VEGF Vascular Endothelial Growth Factor

VSMC Vascular Smooth Muscle Cell

Abstract

An essential event during early pregnancy is the invasion of trophoblasts into the maternal decidua, which is necessary for proper implantation and establishment of maternal-fetal interface and ultimately allows for proper nutrient exchange and immunological tolerance of the growing fetus. For this invasion to occur, cells originating from the trophectoderm undergo an epithelial to mesenchymal transition to become invasive extravillous trophoblasts and begin invading the uterine decidual tissue. Through the secretion of matrix metalloproteinases and through interactions with many cytokines and cell-adhesion molecules, this well-orchestrated process of trophoblast invasion results in extensive remodeling of the maternal spiral vasculature by the extravillous trophoblasts. Ultimately, the spiral arteries are transformed from high resistance, low flow vessels to low resistance, high flow vessels to allow for adequate perfusion of the placenta and developing fetus.

Preeclampsia is a leading cause of maternal morbidity worldwide and is associated with the onset of hypertension and proteinuria, typically after 20 weeks of gestation. While the hypertension typically resolves following delivery of the fetus and placenta, both the mother and growing child are faced with long-term adverse health effects such as the development of cardiovascular disease and metabolic disorders. Preeclampsia is characterized by widespread maternal inflammation and endothelial dysfunction triggered by the secretion of soluble factors from the placenta into the maternal circulation. It is thought that the onset of these adverse systemic conditions is initiated by poor placental

xi perfusion and pathologically hypoxic conditions in the placenta. In many cases of preeclampsia, there is evidence of shallow trophoblast invasion which results in incomplete spiral artery transformation, ultimately leading to poor placental perfusion.

However, the exact mechanisms underlying the inadequate extravillous trophoblast invasion and remodeling are incompletely understood.

LIN28 is an RNA binding protein that is highly expressed in embryonic stem cells, fetal tissues and many cancers, and was discovered as a regulator of the maturation of the Let-7 family of miRNAs. However, as an RNA binding protein, LIN28 has been shown to interact with thousands of mRNA transcripts, leading to both increased and decreased protein expression, and control of many cellular processes such as differentiation, proliferation, migration, invasion, and cellular metabolism. In vertebrates, LIN28 exists as two highly homologous paralogs, LIN28A and LIN28B, however LIN28B is slightly larger and contains a nuclear localization signal not found in LIN28A. While they both function to inhibit Let-7 maturation, there is evidence to suggest they also have independent functions.

Given the primary role of LIN28A and LIN28B in modulating cell metabolism, differentiation and invasion, we hypothesized that LIN28A and/or LIN28B regulates trophoblast differentiation and invasion, and that its dysregulation may contribute to preeclampsia. We found that LIN28B mRNA expression is ~1300-fold higher than LIN28A in human term placenta and is the predominant paralog expressed in human primary cytotrophoblasts, syncytiotrophoblasts, and decidual cells. We also found that LIN28B mRNA and protein levels are significantly reduced in human placentas from preeclamptic pregnancies compared to placentas from normal pregnancies, while LIN28A expression

xii is unchanged. Upon investigation of human first trimester placenta sections, we found that LIN28B is more highly expressed in the invasive extravillous trophoblasts and syncytial sprouts compared to villous trophoblasts. To support this with in vitro evidence, we found that overexpression of LIN28B in human HTR8/SVneo cells resulted in increased proliferation, migration and invasion, while knockdown of LIN28B in JEG3 cells resulted in decreased proliferation. Furthermore, knockdown of LIN28B in JEG3 cells led to decreased expression of SYN-1, ELABELA, and the chromosome 19 miRNA cluster, with accompanying increases in the pro-inflammatory cytokine TNFα and ITGβ4, an integrin enriched on non-invasive trophoblasts. Moreover, culture of JEG3 and BEWO cells in hypoxia resulted in significantly decreased levels of LIN28B mRNA and protein expression, as well as syncytin-1 and ELABELA mRNA levels, while TNFα was increased. These results provide the first evidence that LIN28B is the predominant paralog expressed in human placenta and decreased LIN28B may play a crucial role in

PE pathogenesis by reducing trophoblast invasion, syncytialization and by promoting inflammation.

xiii

Chapter 1

Trophoblast Differentiation and Preeclampsia

Preeclampsia (PE)

Preeclampsia is a severe disease of pregnancy with clinical manifestations that include systolic blood pressure >140mmHg or diastolic blood pressure >90mmHg, accompanied by proteinuria >300mg/day, and is typically diagnosed after 20 weeks of gestation (1). Pathological manifestations of this disease include systemic maternal endothelial dysfunction which results in widespread vasoconstriction as well as elevated vascular permeability (2). Worldwide, PE represents the second most common cause of maternal morbidity, affecting between 3% and 10% of all pregnancies, with mounting evidence indicating a significant increase in the incidence of PE over the last 20 years (3-

5). Furthermore, up to 25% of cases of intrauterine growth restriction (IUGR) and up to

20% of preterm births are attributed to PE (6). Although the exact etiology of PE is poorly understood, it is speculated to arise due to either 1) normal placental-derived factors interacting with maternal conditions that are predisposed or susceptible to cardiovascular disease (maternally driven PE) (7); or 2) the exposure of the placenta to progressive, long-term hypoperfusion and hypoxic conditions, resulting in the release of anti- angiogenic and pro-inflammatory soluble placental factors (placentally driven PE) (8).

Although clinical manifestations of PE do not typically arise until after 20 weeks of

1 gestation, it is thought that PE begins developing early in the pregnancy, well before any clinical adverse effects are detected.

Risk factors associated with preeclampsia

There are many risk factors and predispositions associated with the development of PE. It has been shown that advanced maternal age (i.e. maternal age >35) is a risk factor for PE, although it has also been shown that the incidence of PE is increasing faster amongst women of younger age when compared to older age (9, 10). Furthermore, nulliparous pregnancies are more strongly associated with PE when compared to multiparous pregnancies, possibly due to higher levels of circulating soluble fms-like tyrosine kinase-1 (sFLT-1) in the maternal serum (11). In addition to increased maternal age and nulliparous pregnancies, elevated BMI, African-American race, and the presence of gestational diabetes are frequently associated with the onset of PE (12-15). Lastly, maternal infections and the resulting inflammation is associated with an increased risk for the development of PE. For instance, urinary tract infections and periodontal disease, which can be caused by many different bacterial microorganisms, are strongly associated with the onset of PE (16, 17). In addition, numerous maternal viral infections, such as

CMV, HIV, and HSV-2, have been linked to the onset of PE (18, 19). It is thought that high levels of proinflammatory cytokines as a result of these different infections plays a part in increased oxidative stress and increased circulating anti-angiogenic proteins, thus contributing to the maternal endothelial dysfunction associated with PE (18). It is important to keep these predispositions in mind when studying the underlying development and etiology of PE.

Long- and short-term adverse health effects associated with preeclampsia

To date, the only curative treatment for PE is termination of the pregnancy and/or delivery of the fetus and placenta. Although the widespread maternal endothelial dysfunction and hypertension typically resolve within approximately one month following delivery, short-term and long-term adverse health outcomes exist, for both the mother and child (20). For instance, fetuses exposed to PE in utero are often born early and/or small for gestational age. Furthermore, long-term population-based follow-up studies indicate that children exposed to PE are at a significantly higher risk for the development of long-term cardiovascular, endocrine, and metabolic diseases, such as diabetes and hypertension (21-23). On the other hand, short-term adverse health effects on the mother include antenatal stroke, deteriorated kidney function, elevated liver enzymes, severe headaches and visual disturbances, and elevated circulating C-reactive protein, which indicates the presence of systemic inflammation (24-26). In addition, woman who develop

PE are four-fold more likely to develop cardiovascular disease throughout their lifetime when compared to woman with similar pregnancy numbers and no history of PE (27).

Given these long-term adverse outcomes associated with a disease of this prevalence, it is imperative that the etiology of the disease is further understood, and better treatment options are developed.

Decidualization

Decidualization, beginning after ovulation triggered by high levels of progesterone, is the process in which the decidua is formed and involves extensive morphological and biochemical changes within the endometrium in preparation of implantation of the

3 developing blastocyst (28). Prior to formation of the decidua, endometrial stromal cells

(EnSCs) appear fibroblast like, with little cytoplasm and elongated nuclei. During decidualization, EnSCs adopt a more round nuclei structure and begin to swell due to accumulation of lipid droplets and glycogen in the cytoplasm (29). In addition to morphological changes within the EnSCs, the extracellular matrix is extensively remodeled to facilitate implantation and invasion by trophoblasts. The extracellular composition of collagen IV and laminin is greatly increased during decidualization, and eventually this basement membrane surrounds EnSCs, which facilitates interactions between the decidua and trophoblasts (30). To complement the increase in dense extracellular matrix and basement membrane, EnSCs in the decidua begin secretion of prolactin and IGFBP-1, which are important for modulation of immune cell infiltration and trophoblast invasion (31, 32). In addition to the extracellular matrix composition changes, the mature decidua is characterized by the presence of a large population of decidual natural killer (dNKs) cells around the spiral arteries and endometrial glands (28). Unlike peripheral blood natural killer cells, these dNKs are not cytotoxic and do not typically kill trophoblasts (33). Rather, the dNKs function to secrete cytokines and other factors, such as MMPs, that assist in the disruption of the vascular extracellular matrix and vascular cell-cell interactions that assists in vascular trophoblast invasion, a process that will be discussed in more detail later. It is important to note that decidual cells (DCs) express vimentin, but not cytokeratin, as opposed to invasive trophoblasts which express cytokeratin, but not vimentin, thus making the two cell types easily distinguishable using immunohistochemical staining (34-36).

Development of the trophectoderm and inner cell mass

The embryo begins as a totipotent, single celled organism following fusion of an oocyte and sperm. After successful fusion, the first differentiation event of embryogenesis and multiple cycles of cell division occur, which ultimately results in two distinct populations which arise due to asymmetrical division during the transition from the 8- to

16-cell stage. Blastomeres on the outer surface of the embryo display distinct polarity and gene expression, while blastomeres on the inner portion of the embryo lack polarity and display an entirely separate distinct set of genes (37, 38). The polarized blastomeres on the outer surface of the embryo are destined to differentiate into the trophectoderm (TE), while the blastomeres on the inner portion of the embryo become the pluripotent inner cell mass (ICM), and eventually, the three germ layers (Figure 1). The differential gene expression between these two layers is achieved through many different mechanisms, such as histone modification, DNA methylation, and differential miRNA expression (39-

41). Currently, the exact time in which developmental bias towards the TE or the ICM remains controversial, with some reports indicating this begins as early as the 2-cell stage, and others indicting this begins around the 8-cell stage (38). Due to ethical concerns, the study of human ICM and TE formation and function has proven difficult.

Yet, despite some species-to-species variation, a wealth of knowledge has been accrued based on studies of mouse tissues.

The ICM is a pluripotent cluster of cells within the primordial embryo which will eventually differentiate into the three germ layers and, ultimately, the developing fetus.

One of the first factors identified in formation and maintenance of the ICM was the transcription factor Oct4. Oct4 is a transcription factor that is expressed substantially

5 higher in the ICM compared to the TE, and regulates the expression of FGF-4. Oct4 induced expression of FGF-4 is highly important, as FGF-4 is secreted from the ICM in a paracrine manner and stimulates growth and proliferation of the developing TE (42).

Furthermore, knockout of Oct4 is embryonically lethal and results in an ICM that is not pluripotent and constrained to differentiation along the trophoblast lineage (42). Nanog is another transcription factor that regulates formation of the ICM. In a manner similar to

Oct4, Nanog’s expression is restricted to the ICM, and Nanog-null embryos fail to develop an epiblast, suggesting a lack of pluripotency of the ICM (43). Furthermore, culturing of

Nanog-deficient embryonic stem cells (ESCs) results in spontaneous differentiation into the extraembryonic lineage, further indicating that Nanog is essential for maintenance of pluripotency of the ICM (43). Another transcription factor, SOX2, is also required for proper development and pluripotency of the ICM. Much like Oct4, isolated cells from

Sox2-null ICM spontaneously differentiate along the trophoblast lineage, suggesting it is also vital for the ICM (44). Sox2 forms a heterodimer with Oct4, and together they regulate the expression of many genes that protect the pluripotency of the ESCs, while disruption of this interaction leads to differentiation into the primitive endoderm (45, 46). The careful balance of these important transcription factors, among others, within the ICM not only maintains pluripotency, but also directs cell differentiation along specific lineages to allow for proper development of the three germ layers and the fetus (47, 48).

Polar epithelial cells of the TE differentiate into trophoblasts, which form the building blocks of the placenta, and are indispensable for initial implantation of the blastocyst in the uterine wall. Development of the TE, and later differentiation, requires a set of genes which are distinct from that of the ICM (38, 48). For instance, the

6 transcription factor Tead4 is highly expressed in the TE, and Tead4 knockouts display lethality prior to implantation (49, 50). Tead4 is responsible for the activation of many downstream genes that are required for proper development of the TE, such as the FGF receptor and the transcription factor Cdx2. Like Tead4, Cdx2 is highly expressed in the

TE, and is required for its development, as Cdx2-null blastocysts fail to develop a proper

TE and fail to implant (51). Furthermore, Cdx2-null blastocysts do not develop trophoblast stem cells, whereas forced expression of Cdx2 in ESCs results in conversion to trophoblast stem cells (52). Through the activation of other genes and transcription factors, Cdx2 promotes symmetrical cell division during early blastocyst formation and promotes cell polarity, which is crucial for proper TE development. Interestingly, Cdx2 has also been shown to occupy the promoter region of Oct4 in order to prevent its expression within the TE layer (38). Tight regulation of these genes, among many others, is required for the proper development of the TE and, ultimately, implantation into the uterine wall and development of the functional placenta.

Figure 1. Simplified schematic representation of blastocyst prior to implantation.

Polar epithelial cells of the trophectoderm, which differentiate into trophoblasts and extraembryonal tissues such as the placenta, attach to and invade the maternal endometrium. Conversely, non-polar pluripotent cells of the inner cell mass eventually differentiate into the three germ layers, and ultimately the fetus.

The placenta

The placenta is a vital organ of pregnancy that serves as a bridge between the developing fetus and the maternal uterus. As a key regulator of nutrient uptake, gas exchange, waste elimination, and hormone production, an appropriately developed and functioning placenta is vital for the healthy development and progression of the fetus and pregnancy. In humans, the placenta develops circumferentially with the most extensive invasion at the center and less extensive invasion towards the periphery, which results in a discoid shape upon complete development (53, 54). This invasion, in addition to many other molecular, biochemical, and physiological changes that occur are required to produce favorable conditions for the developing fetus.

Placentation in early pregnancy

Human decidualization begins on day 14 of the menstrual cycle, in preparation for adhesion of the blastocyst to the uterine decidua. Blastocyst adhesion, which takes place approximately one week after fertilization, is the first step in implantation, and triggers the development of the maternal-fetal junctional zone (55). Implantation and formation of the maternal-fetal junctional zone triggers trophoblast proliferation and differentiation into syncytiotrophoblasts (STs), as well as penetration of the uterine epithelium and which triggers erosion of maternal capillaries and glands, allowing blood and gland contents to enter the lacunae (56). This process also initiates trophoblast differentiation into villous cytotrophoblasts (CTs), which proliferate to form primary, secondary, and tertiary villi, or extravillous trophoblasts (EVTs), which further differentiate into interstitial EVTs or endovascular EVTs. EVTs have the capacity to degrade extracellular matrix, allowing

9 them to invade the maternal decidua and remodel the maternal spiral arteries (57, 58).

Furthermore, non-invasive CTs on the anchoring villi appear to express integrinβ4, whereas invasive EVTs express integrinβ1, indicating that the differentiation from non- invasive to invasive trophoblasts involves changes in cell-surface integrins (59-61).

The uterine vasculature undergoes extensive structural and physical changes during gestation to provide adequate blood flow and nutritional support to the uterus, placenta, and growing fetus. These changes, along with formation of the placenta and implantation process (i.e. placentation) during early pregnancy, are a very complex and intricately orchestrated chain-of-events that ultimately results in the development of adequate utero-placental perfusion. While all of the details underlying this process are not completely understood, it is well established that early in the pregnancy, approximately 5-8 weeks after implantation, fetally derived trophoblasts undergo an epithelial-to-mesenchymal transition (EMT) to become EVTs and begin invading the maternal tissue, a process which is typically completed by 20 weeks of gestation (62, 63).

The invasion of EVTs into maternal tissues is categorized as vascular invasion (invasion of the EVTs resulting in remodeling of the maternal spiral arteries) and interstitial invasion

(invasion of the EVTs to the space between arteries). Together, this invasion of the maternal tissue paves the way to adequate placental perfusion.

Interstitial EVT invasion

Interstitial trophoblast invasion involves invasion of EVTs from anchoring villi into the decidual tissue located between maternal spiral arteries. Through the release of

MMPs, such as MMP-2, -9, and -12, it is thought that these interstitial trophoblasts begin

10 degrading extracellular matrix and promote further invasion. In addition to the release of

MMPs, activation and recruitment of growth factors, cell-surface receptors, and cytokines such as IL-8 and IL-13, by EVTs further stimulates the process of proper implantation and development (64-66). In fact, it has recently been shown that MMP-12 has elastolytic capacities, which assists in EVT remodeling of the spiral arteries (67). Under normal circumstances, the interstitial invasion of the EVTs eventually reaches the myometrial area and terminates with the fusion of EVTs to form multinucleated giant cells (68). In parallel to the interstitial trophoblast invasion and degradation of the decidual extracellular matrix, invasive EVTs also invade and remodel the maternal spiral arteries.

Vascular EVT invasion

The spiral artery vascular invasion by EVTs is a process in which high-resistance, small diameter spiral arteries are remodeled and transformed into low-resistance, large diameter vessels with increased flow (69). This intricate process is initiated by the invasion of EVTs into the distal portions of the uterine arteriole, followed by retrograde invasion and migration until reaching the spiral artery (70). Upon reaching the lumen of the spiral artery, these endovascular trophoblasts accumulate, resulting in the formation of the trophoblast plug (71). These trophoblast plugs within the maternal spiral arteries prevent blood flow from reaching the developing placenta and fetus during the early weeks of pregnancy. However, to allow for proper nutrient delivery, invasive extravillous trophoblasts also invade the uterine glands, eventually eroding the epithelium and allowing their contents to be secreted into the intervillous space, thus providing adequate nutrient flow prior to complete spiral artery remodeling throughout the first ~10-12 weeks

11 of gestation (72, 73). By the 12th week of gestation, the trophoblast plugs within the spiral arteries loosen and degrade, which allows for the flow of maternal blood into the intervillous space of the developing placenta (74). However, the remodeling of the maternal spiral arteries does not stop with the degradation of the trophoblast plugs. The

EVTs continue to migrate and invade in a retrograde fashion until the myometrial portion of the spiral artery. During this invasion, there is a downregulation of surface adhesion receptors, such as E-cadherin, on the EVTs, while there is an upregulation of VE- cadherin, VECAM-1, and PECAM-1, which facilitates invasion and migration of the endothelial cell layer of the spiral arteries (55, 75, 76). Along with EVT invasion of the spiral arteries comes vascular endothelial cell apoptosis, followed by the accumulation of trophoblasts, thus forming a new trophoblast cell endothelial-like barrier lining the lumen of the spiral artery (77). It is thought that the apoptotic endothelial cells are rapidly removed by phagocytosis to prevent the secretion of immunogenic contents from within the dying cell (70).

In addition to the removal and replacement of endothelial cells by trophoblasts during spiral artery remodeling, vascular smooth muscle cells (VSMCs) are also altered.

Under typical conditions, VSMCs exist in the functional, contractile phenotype. However, it is well known that they can switch from this contractile phenotype to the synthetic, proliferative phenotype (78, 79). During spiral artery remodeling, loss of the VSMC contractile phenotype is thought to be associated with the loss of the layered structural organization of the medial layer of the spiral artery. In addition to this loss of VSMC organization, trophoblast invasion also results in VSMC apoptosis and replacement of the

VSMCs with fibrinoid deposits and trophoblasts (80). It is important to note that, while the

12 vast majority of vascular remodeling takes place in the decidual spiral arteries, the decidual veins and lymphatics are also remodeled, albeit to a much lesser extent. It is thought that this remodeling serves to increase the maternal-fetal interface, thus allowing increased nutrient exchange (81). Overall, the complex remodeling of the decidual vasculature results in a substantial increase in the diameter of the lumen and diminished contractile capabilities of the arteries, thus allowing adequate maternal blood flow into the intervillous space to support the growing placenta and embryo. However, in PE affected placentas, this remodeling is often impaired, resulting in inadequate blood flow and prolonged, progressive hypoperfusion and hypoxic conditions for the developing fetus.

Syncytialization

Trophoblast syncytialization is the process in which CTs fuse to form a continuous layer of multinucleated cells, referred to as the syncytium. Primary trophoblast syncytialization occurs at the time of implantation when the blastocyst first comes into contact with maternal decidua and serves as the basis for the development of the syncytium. This initial lateral fusion of neighboring trophoblasts allows for proper attachment and implantation, resulting in degradation of the maternal capillaries and glands which allows blood to enter the newly formed lacunae (56). To aid in growth of the evolving syncytial layer, CTs distal to the STs continue to proliferate and fuse with the

STs, which leads to the formation of projections that result in areas of empty space and trabeculae, which eventually become the placental villi. This initial attachment and primary syncytialization culminates around post fertilization day 11 when the blastocyst is

13 fully implanted (82). On the other hand, secondary syncytialization occurs continuously throughout the duration of the pregnancy to maintain placental epithelial barrier integrity.

Secondary syncytialization results in formation of the mature syncytium which exists as a functional border between the maternal circulation and the developing fetus.

The syncytium is the principal site of maternal-fetal gas and nutrient exchange, as well as waste elimination. Health and function of the syncytium is maintained by continual cellular replenishment by fusion of villous CTs into the ST layer, which involves the transfer of the nucleus and cytoplasm of the CTs into the syncytium (83). The intricate balance of the syncytium is maintained by continual shedding of syncytial cells and apoptotic material into the maternal circulation to allow for the constant addition of newly fused CTs (82, 84,

85). Proper formation and maintenance of the syncytium is vital, and dysregulation can result in pregnancy failure or abnormal outcomes such as PE or IUGR (86, 87).

Fusion of CTs to the syncytial layer of the placental villi requires a complex cascade of events that includes membrane destabilization, expression of cell fusogens, and ultimately the uptake of cellular contents of the fusing CT into the syncytium. While the exact signaling mechanisms responsible for proper fusion remain an area of active investigation, it is well established that SYN-1 plays a vital role in this process. SYN-1 is an envelope protein belonging to the human endogenous retrovirus family W, which is highly expressed in STs of the placenta and helps mediate the fusion of CTs into the syncytium (88). Many signaling cascades have been implicated in the expression of SYN-

1, with most working through the activation of GCM-1, a transcription factor responsible for the expression of SYN-1, and to a lesser extent the related protein SYN-2 (89-91). For instance, cAMP/PKA dependent post-translational modifications of GCM-1 results in

14 stabilization of the protein and enhanced expression of SYN-1 (90). Furthermore, in vitro inhibition of ERK1/2 or p38MAPK results in reduced syncytialization, while activation of p38MAPK has been shown to induce SYN-1 expression through activation of GCM-1 (92,

93). Lastly, it has recently been shown that activation of the β-catenin pathway results in the upregulation of GCM-1, which in turn leads to the induced expression of SYN-1 and syncytialization (94). Overall, the development and maintenance of a functional syncytium is necessary for the healthy progression of the pregnancy and impaired syncytium formation may result in pregnancy related disorders such as PE.

Impaired vascular remodeling in preeclampsia

Shallow invasion and impaired vascular remodeling of the maternal spiral arteries

(i.e. impaired placentation) has been well established as an underlying pathology of PE

(95, 96). In contrast to the extensive remodeling that is observed in normal pregnancies, spiral artery transformation appears to be incomplete in PE, with fewer endovascular trophoblasts and more abundant spiral arteries that retain an intact endothelial and VSMC layer (Figure 2) (97). Furthermore, it has been shown that invasive EVTs from PE affected placentas display reduced VE-cadherin and integrin α1, which potentially contributes to the reduced endovascular invasion evident in the disease (98). Due to the inadequate remodeling, the spiral arteries fail to sufficiently dilate, thus resulting in inadequate delivery of maternal blood to the placenta and exposure of the placenta to increasingly hypo-perfused and hypoxic conditions. As a result of these hypoxic conditions, there is a marked downregulation of syncytialization mediator SYN-1, leading to reduced syncytiotrophoblast formation, which is evident in placentas complicated by

PE (99-101). It is important to note that, while abundantly evident in PE, shallow implantation is implicated in other pathological conditions of pregnancy, such as IUGR and some cases of pre-term birth (102, 103). In addition to the physical changes in the placenta induced by the aberrant invasion and remodeling of the spiral arteries, the long- term hypo-perfusion and hypoxia has been shown to induce secretion of soluble factors into the maternal circulation that promote inflammation and widespread endothelial cell dysfunction (104, 105). Currently, the underlying molecular and biochemical aberrations that cause impaired invasion and vascular remodeling in PE is currently unknown.

Figure 2. Simplified schematic showing spiral artery remodeling by invasive EVTs.

In diseases such as PE, impaired spiral artery remodeling by invasive EVTs results in maintained spiral artery vascular tone and decreased maternal blood flow to the developing fetus and placenta (left). In contrast, adequate vascular remodeling (right) by invasive EVTs results in increased lumen diameter and loss of contractile phenotype allowing for sufficient maternal blood flow to the placenta and developing fetus.

17 sFLT-1 and preeclampsia

Vascular endothelial growth factor (VEGF) is known to be a promoter of angiogenesis and plays a role in regulating blood pressure and proteinuria. Circulating

VEGF can induce endothelial dependent vasodilation through the AKT dependent phosphorylation and activation of endothelial nitric oxide synthase (eNOS), as well as induction of prostacyclins, thus decreasing blood pressure (106). Conversely, VEGF signaling inhibitors have been shown to induce hypertension though disruption of endothelial dependent vasodilation, and proteinuria, through disruption of the glomerular barrier integrity (107, 108). sFLT-1 is a soluble splice variant of the VEGF receptor FLT-

1, which lacks the transmembrane and cytoplasmic domain of the receptor. Circulating sFLT-1 can bind and sequester circulating VEGF and placental growth factor (PlGF), thus acting as an inhibitor of VEGF signaling and promoting hypertension and proteinuria

(109). sFLT-1 is expressed by many tissue types, including the placenta, and has been shown to be upregulated in the placenta, the amniotic fluid, and in the maternal circulation of pregnancies complicated by PE, with the circulating levels of sFLT-1 typically normalizing within ~48 hours of delivery (104, 110-112). Additionally, hypoxia has been shown to upregulate sFLT-1 in CTs (113). Furthermore, exogenous overexpression of sFLT-1 in pregnant or non-pregnant rodents results in severe hypertension as well as renal lesions, further implying that sFLT-1, not necessarily the placenta, is responsible for the maternal endothelial dysfunction (104). In the time since these findings, sFLT-1 has been investigated as a biomarker for the prediction of PE early in pregnancy, however sFLT-1 alone is not enough to accurately predict the development of the disease. Rather,

18 the sFLT-1/PlGF ratio has shown promise in clinical studies as a predictive biomarker for early onset PE, although more trials are currently underway (114-116).

Soluble endoglin in preeclampsia

The exogenous delivery of sFLT-1 does not induce hemolysis and thrombocytopenia, which are symptoms of HELLP syndrome associated with severe PE, which indicates that other soluble, placental derived factors may be involved in the disease development. For instance, Endoglin (Eng), a cell-surface receptor for transforming growth factor β1 (TGFβ1) and β3 (TGFβ3), is highly expressed on endothelial cells and STs, and has been shown to associate with eNOS to regulate vascular tone (117-119). Gene expression profiling of placental tissues obtained from normal and PE pregnancies indicate that Eng is upregulated in PE. Furthermore, a soluble form of Endoglin (sEng), is shown to be upregulated in the serum of women with

PE, and its expression level correlates with disease severity (105). In addition to being elevated in the serum of PE affected women, exogenous overexpression of sEng in rodents resulted in hypertension, which was exacerbated by co-overexpression of sFLT-

1 and sEng. Moreover, co-overexpression of sFLT-1 and sEng resulted in severe proteinuria and biochemical evidence of HELLP syndrome (105). Because the increase in sEng can be observed in the serum of women as many as 10 weeks before the onset of PE, it has been posited as a predictive biomarker for the development of the disease.

Together with sFLT-1, sEng plays a major role in contributing to the systemic adverse effects observed in PE.

C19MC in the placenta and preeclampsia

The chromosome 19 microRNA (miRNA) cluster (C19MC) is one of the largest known miRNA clusters, is primate specific, genomically imprinted, and expressed almost exclusively in the placenta (120). The C19MC spans over 100kb of genomic DNA, with its expression resulting in over 40 different mature miRNAs (121). It has been shown that these miRNAs are highly expressed in CTs and STs and can be shed into the maternal circulation through exosome shedding from the STs (122). In fact, the miRNAs from the

C19MC cluster are among the most abundantly detected miRNAs in the placenta and in the serum of pregnant women. Although the complete role of these miRNAs in the placenta is not currently known, it has been shown that the C19MC miRNAs can confer viral resistance to trophoblasts, and that some miRNAs of the cluster modulate the migration, but not proliferation, of trophoblasts (123-125). However, while the role of the

C19MC in the placenta is incompletely understood, it should be noted that the expression of some miRNAs of the C19MC have been shown to be downregulated in the placenta of

PE affected pregnancies, while upregulated in the serum of PE affected women (126,

127).

Inflammation in preeclampsia

Maternal immune tolerance is a key facet of pregnancy. Proper interaction between the maternal immune system and the placenta and developing fetus is required for placentation and success of the pregnancy. However, PE is often associated with aberrant activation of the immune system characterized by increased circulating levels of proinflammatory cytokines. For instance, the proinflammatory cytokine TNFα is highly

20 expressed early in pregnancy and is necessary for proper implantation, while overproduction of this cytokine has been linked with implantation failure and recurrent pregnancy loss (128). Moreover, TNFα has been reported to be upregulated in the placenta, the amniotic fluid, and the maternal circulation in pregnancies affected by PE

(129-131). The increased level of circulating TNFα can contribute to endothelial dysfunction by increasing endothelial cell permeability and increasing the expression of adhesion molecules on the endothelial layer. Aberrant TNFα signaling has been shown to lead to a reduction in NO signaling by reducing the half-life of NOS mRNA, while also increasing the expression of preproendothelin-1, the precursor to the potent vasoconstrictor endothelin-1 (132, 133). This decrease in vasodilatory signaling coupled with an increase in vasoconstriction signaling is an underlying contributing factor to the increase in blood pressure associated with PE.

Chapter 2

The RNA Binding Protein LIN28

Introduction to LIN28

LIN28 is an evolutionarily conserved RNA binding protein that was originally discovered using mutagenesis screens investigating heterochronic genes in C. elegans, where it was shown to play a vital role in the regulation of developmental changes between larval stages (134, 135). Since the time of its discovery, LIN28 has been found to regulate development and pluripotency. For instance, LIN28 is highly expressed in

ESCs and is used in the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), while it is typically downregulated in terminally differentiated cells (136-

139). In C. elegans, LIN28 exists as a single paralog whereas in all vertebrates, LIN28 exists in two paralogs: LIN28A and LIN28B, both of which function as RNA binding proteins and likely arose from an ancestral gene duplication (140, 141). LIN28A and

LIN28B share 65% sequence homology, with the highest degree of homology existing within the cold shock domain (CSD) and cysteine cysteine histidine cysteine zinc knuckle domain (CCHC), two RNA binding domains which are present on both paralogs (134,

142, 143). However, human LIN28B is approximately 41 amino acids larger than LIN28A, and it contains two putative nuclear localization signals: one in the region between the

CSD and the CCHC domains, and one at the extended C-terminus of the protein (Figure

3) (144, 145). Furthermore, LIN28B exists in humans as two isoforms, with one being

22 found predominantly in the placenta and undifferentiated tissues, and the other being found in fetal liver and non-tumor liver tissues (141).

The most well-understood pathway of LIN28A and LIN28B involves their regulation of the Let-7 family of miRNAs. However, it is important to note that, while LIN28A and

LIN28B inhibit the maturation of Let-7, they themselves are also targets of Let-7, a highly conserved cellular regulatory process known as the LIN28/Let-7 bistable switch (Figure

3). Therefore, it is important to keep in mind that in cell lines/tissues in which LIN28A and/or LIN28B expression is high, Let-7 expression is low, and when Let-7 expression is high, LIN28A and LIN28B expression are low.

Figure 3. Schematic representation of LIN28A and LIN28B protein.

A. Human LIN28A and LIN28B both contain the cold shock and the CIS-CIS-CIS-HIS RNA binding domains, however LIN28B is slightly larger than LIN28A. B. In less differentiated tissues, such as ESCs and trophoblasts, LIN28A and LIN28B are abundantly expressed while let-7 expression is reduced, whereas in terminally differentiated cells, LIN28A and LIN28B expression are reduced, and Let-7 is abundantly expressed.

Regulation of Let-7 by LIN28A and LIN28B

The Let-7 family of miRNAs consists of 12 different miRNAs and is a conserved family of tumor suppressors that function to regulate cellular differentiation and proliferation through inhibition of RAS, MYC, and Hmga2 (146, 147). Loss of expression of Let-7 can result in the cell adopting a less differentiated state, such as what is observed in many cancers (148). LIN28A and LIN28B bind an evolutionarily conserved GGAG motif in pre-let-7, thereby preventing its maturation (149). This bistable LIN28/Let-7 switch is by far the most studied cellular function of LIN28 and Let-7. However, it is important to note that LIN28A and LIN28B have also been shown to promote translation of mRNA, which will be further discussed later (150-152). While LIN28A and LIN28B are structurally very similar, they function to inhibit the maturation of Let-7 by distinct mechanisms.

LIN28A has been shown to inhibit the maturation of both pri-Let-7 and pre-Let-7 transcripts. LIN28A can function in the nucleus by co-transcriptionally binding pri-Let-7 miRNA transcripts. This binding of LIN28A to the primary transcript is thought to physically prevent further processing by DROSHA, and therefore preventing maturation of Let-7

(153). More abundantly, LIN28A can function in the cytoplasm to inhibit the maturation of pre-Let-7 transcripts. The recognition and inhibition of maturation of pre-Let-7 requires both the CSD and the CCHC domain of LIN28A (144). Through interactions of these domains with the terminal loop of precursor Let-7 transcripts, LIN28A recruits the terminal uridylyltransferase (TUTase4/Zcchc11 and/or TUTase7/Zcchc6) which results in oligouridylation of the pre-Let-7 transcript (Figure 4). The oligouridylation of pre-Let-7 leads to rapid degradation of the miRNA transcript, thereby preventing the final maturation by DICER (154-156).

LIN28B is typically expressed in the nucleus due to the functional nuclear localization signal on the C-terminal end of the protein. While some in vitro studies have shown that LIN28B has the capacity to recruit the uridylyltransferase Zcchc11 and

Zcchc6, knockdown of Zcchc11 in cell lines that express only LIN28B, and not LIN28A, does not result in an increase in Let-7 expression (145, 154). Furthermore, Zcchc11 is typically found only in the cytoplasm, which further indicates that LIN28B exerts its inhibitory regulation of Let-7 through a different mechanism than LIN28A (157). In fact,

RNA immunoprecipitation and immunohistochemical analyses of LIN28B indicates that

LIN28B has a very high affinity for Let-7 primary transcripts and that it binds and sequesters the primary transcripts in the nucleolus, where the miRNA processing machinery is not present (Figure 4) (145).

Figure 4. Mechanism of Let-7 repression by LIN28A and LIN28B.

LIN28B binds to primary Let-7 transcripts and sequesters them in the nucleolus, away from the miRNA processing machinery. Conversely, LIN28A binds pre-Let-7 miRNA transcripts in the cytoplasm to prevent maturation by DICER and associates with TUTase4 and TUTase7 to polyuridylate and tag the transcript for degradation.

Gene regulation by LIN28A and LIN28B independent of Let-7

While regulation of Let-7 by LIN28A and LIN28B is very important and well- understood, it is important to note their Let-7 independent functions. PAR-clip analyses have revealed that LIN28A and LIN28B bind over 3,000 mRNA transcripts, and this interaction leads to a moderate increase in the protein abundance (158). Furthermore,

CLIP-seq analyses of ESCs and human somatic cells stably transfected with LIN28 reveal that LIN28 binds to mRNA transcripts at GGAGA motifs, directly regulates the protein expression level of RNA binding proteins, and regulates splicing factor abundance, with changes in LIN28 levels resulting in widespread changes in alternative splicing (159). In addition to the GGAGA motif, it has been shown that LIN28A and LIN28B recognize an

‘A’ bulge, which is flanked by two G:A base pairs on mRNA transcripts. Recognition of this motif, which is embedded in the secondary structure of the mRNA transcript, results in association with LIN28A or LIN28B and increased translation of the transcript (160).

However, it has also been shown that LIN28A, through recognition of conserved motifs in the stem loop of some mRNA secondary structures, can repress translation of mRNAs that are destined for the endoplasmic reticulum, thus reducing the translation of transmembrane and secreted proteins (161).

While many different gene types are targeted by LIN28A and LIN28B, a large portion of the genes are related to cell-cycle progression and promote cellular growth and survival. In addition to regulation of alternative splicing and splicing factor abundance,

LIN28A has been shown to directly bind to the mRNA transcript of IGF-2 in skeletal muscle precursors, resulting in enhanced translation of the IGF-2 protein (152). It has previously been shown that LIN28A and LIN28B can recruit RNA helicase A, which,

28 through interactions with the C-terminus of LIN28, assists in the remodeling of ribonucleoprotein interactions leading to increased translation, which could mechanistically explain the LIN28A and LIN28B induced increase in protein translation

(150). In fact, deletion of the C-terminal end LIN28, while leaving the RNA binding domain intact, results in decreased protein translation (150). Furthermore, it is important to note that LIN28B protein has been shown to bind and enhance translation of its own mRNA, thus further increasing its own expression level (158, 159). This feed-forward mechanism plays a part in maintaining high levels of LIN28B, further promoting genes associated with cell-cycle progression and cellular growth. However, further studies are warranted to validate the abundant targets of LIN28A and LIN28B, and to determine their precise role in normal and pathological conditions.

Elegant studies using transgenic and knockout animals have provided more insight into Let-7 independent gene regulation by LIN28A and LIN28B. For instance, mice with a skeletal muscle specific deletion of LIN28A display insulin resistance and impaired glucose tolerance, notwithstanding any changes in Let-7 expression, while transgenic mice overexpressing LIN28A are larger and have increased organ sized despite no change in Let-7 expression in the respective organs (162, 163). These studies, along with numerous other in vitro and in vivo studies, indicate that LIN28A and LIN28B play a major role in growth and metabolism.

Regulation of cellular growth and metabolism by LIN28A and LIN28B

In recent years it has become well-established that LIN28A and LIN28B play a major role in cellular metabolism, as well as proliferative growth. For instance, repression

29 of Let-7 by LIN28A and LIN28B has been shown to enhance mTOR signaling, resulting in increased biogenesis of ribosomes and increased translation, while LIN28A has been shown to directly bind the transcripts of many ribosomal peptides, further increasing protein abundance and cellular growth (164-166). Also, through the repression of Let-7,

LIN28A and LIN28B can upregulate the RAS, Myc, and PI3K-AKT pathway, all of which result in increased cellular metabolism (162, 167). Furthermore, and again through the regulation of Let-7, LIN28A and LIN28B can increase the expression of Hmga2, which represses the cyclin dependent kinase inhibitor p16, and thus, promotes cellular proliferation and survival (168, 169). Moreover, through regulation of the PI3K-mTOR pathway, mice genetically engineered to overexpress LIN28A or LIN28B have increased body size and length, in addition to increased glucose metabolism and insulin sensitivity accompanied by a resistance to obesity and diabetes (162, 163). Taken together, it is clear that both LIN28A and LIN28B, whether directly or indirectly through its regulation of the Let-7 family of miRNAs, plays a pivotal role in promoting cellular growth, proliferation, and metabolism.

Upstream regulation of LIN28A and LIN28B

The regulation of LIN28A and LIN28B by the Let-7 family of miRNAs is well characterized, however relatively little is known about the upstream transcriptional control of their expression. The upstream regions of LIN28A and LIN28B have many predicted transcription factor binding sites, although the vast majority have yet to be characterized and validated in vitro. High resolution CHIP-seq analyses have revealed that the pluripotency transcription factors Sox2, Nanog, Oct4, and Tcf3 associate with the

30 promoter region of LIN28A, thus driving its expression (170). In addition to promoting the transcriptional expression of LIN28A, Sox2 can form protein-protein interactions with

LIN28A in the nucleus, a process in which further promotes pluripotency (171, 172).

LIN28A and LIN28B have also been shown to be upregulated in response to TGFβ, potentially through the repression of histone methyltransferases and/or the upregulation of Smad proteins which interact with a Smad binding element in the promoter region of

LIN28B, thus driving its expression (173, 174). Furthermore, bioinformatic analysis reveals a conserved NF-κB binding element within the LIN28B gene, which results in rapid induction of LIN28B expression following activation of NF-κB signaling (175). Lastly,

CHIP-seq analyses have revealed that the growth factor Myc binds to the upstream promoter region of LIN28B, resulting in robust expression (176). It is important to note that, while regulating many similar functions such as cellular growth, metabolism, and reprogramming, LIN28A and LIN28B appear to be transcriptionally regulated by distinct mechanisms. Additional studies are warranted to further elucidate the precise upstream regulatory mechanisms of these genes.

LIN28A and LIN28B in embryogenesis and pluripotent stem cells

During early embryogenesis, LIN28A protein expression is attained through maternal inheritance from the oocyte (177). In the mouse, LIN28A is highly expressed in nucleoli precursors of the 2-, 4-, and 8-cell stage, and is very highly expressed in the mature nucleoli of the blastocyst. However, in non-human primate embryogenesis,

LIN28A is observed to be expressed in the nucleoli of the outer blastomeres, suggesting a different pattern of expression amongst species. Nevertheless, this compartmentalized

31 expression is speculated to regulate the maturation of nucleoli prior to the activation of the embryonic genome (177, 178). Upon activation of the embryonic genome, both

LIN28A and LIN28B are highly expressed in the inner cell mass, which is also true for in vitro cultured ESCs. Furthermore, RNAi mediated knockdown of LIN28A in ESCs results in defects in the cells proliferative capacity, while overexpression increased the rate of cell division and accelerated the formation of iPSCs (137, 179). However, considering

LIN28A or LIN28B knockout mice develop through the blastocyst stage relatively normally in utero, it appears that LIN28A and/or LIN28B are not absolutely required for pluripotency, at least in vivo (180). Taken together, these data indicate that LIN28A and

LIN28B play a vital role in early embryonic development, as well as in the maintenance of pluripotent stem cells.

LIN28A and LIN28B in cancer and cellular invasion

Many of the proliferative, migratory, and invasive pathways that are activated during normal embryogenesis and trophoblast invasion during pregnancy are exploited by cancer cells during growth and metastasis (181, 182). Considering the major role of

LIN28A and LIN28B in the regulation of cellular growth, proliferation, metabolism, and stemness, it is no surprise that the reactivation of LIN28A and/or LIN28B and subsequent loss of Let-7 has been reported in many cancers including breast, cervical, colon, esophageal, gastric, and liver, amongst many others (183-186). Furthermore, the degree of LIN28A and/or LIN28B reactivation and Let-7 loss is highly correlated with disease progression and poor prognosis (187). It has been postulated that the increased

LIN28A/LIN28B signaling in these cancers results in high expression levels of many

32 oncogenes, including K-RAS, c-Myc, and Hmga2, through repression of Let-7 signaling.

In addition, high levels of LIN28A and LIN28B have been observed in cancer stem cells and have been shown to enhance the formation of these cells, further implicating LIN28 in disease progression (188, 189).

Prior to EVT invasion into the maternal decidua, placental trophoblasts undergo an EMT, resulting in the ability to migrate and invade (190). In a similar manner, cancers cells must undergo an epithelial to mesenchymal transition prior to local invasion of surrounding tissue in a process that has been shown to be induced by expression of

LIN28A (188). EMT is characterized by the loss of E-cadherin expression with a parallel increase in mesenchymal transcription factors such as Snail, Slug, and Twist. In fact, many studies of multiple cancer types have shown that LIN28A and LIN28B promotes cancer invasion through promotion of EMT, while Let-7 inhibits invasion (191). LIN28A and LIN28B promote invasion through the repression of Let-7, which in turn leads to an increase in the expression of Hmga2, which promotes EMT through the upregulation of

Snail and Slug and downregulation of E-cadherin (192). Furthermore, LIN28A and

LIN28B have been shown to interact with and promote the translation of Hmga1, another factor which also promotes EMT, while also interacting with E-cadherin mRNA and repressing its translation (161, 165, 193). Based on these findings, it is clear that LIN28A and LIN28B play a major role in EMT and cancer cell invasion in a process that parallels that of initial trophoblast invasion during pregnancy.

Gap in knowledge and purpose of this dissertation

LIN28A and LIN28B are RNA binding proteins that are highly expressed in ESCs and other less differentiated cells and were initially discovered as regulators of Let-7 miRNA maturation. However, LIN28A and LIN28B are also expressed in the developing embryo, fetal tissues, the testes, the placenta, and in cancer cells. In the years since their discovery, it has been shown that, in addition to the regulation of Let-7, these proteins play a major role in mRNA translation and the regulation of splicing factor abundance.

Through these interactions with many different miRNAs and mRNAs, LIN28A and LIN28B function to regulate numerous different cellular processes, such as cellular metabolism, growth, proliferation, and invasion.

PE is a disease of pregnancy that is characterized by the onset of hypertension and proteinuria after 20 weeks of gestation. In addition to the short-term unfavorable maternal health effects caused by PE, there are many long-term adverse outcomes that affect both the mother and child. Epidemiological studies have indicated that long-term maternal adverse outcomes include prolonged hypertension and kidney disease. On the other hand, long-term health problems for the child include a much greater likelihood of developing hypertension, kidney disease, and metabolic disorders such as diabetes.

Although the clinical manifestations of PE do not typically arise until >20 weeks gestation, the underlying pathological changes that lead to the development of PE are thought to begin much earlier.

Early in pregnancy, upon implantation of the blastocyst to the maternal decidua, invasive EVTs begin invading and remodeling the maternal spiral arteries. This remodeling results in a phenotypic change of the arteries from a high pressure, low flow

34 system, to a low pressure, high flow system that allow for deliver adequate blood to the developing placenta and fetus. In PE, due to currently unknown molecular and biochemical aberrations, this invasion and remodeling is inadequate, which results in the placenta and fetus being exposed to prolonged increasingly hypoxic conditions.

Despite the enormous amount of knowledge regarding LIN28, it is not currently known which paralog is primarily expressed in the human placenta, and in which placental cell types it is expressed. Furthermore, the regulation and pathophysiologic role of

LIN28A and/or LIN28B in placenta associated disorders, such as PE, has not been investigated. To shed light on the role of LIN28A and LIN28B in the placenta in view of its predominant placental expression and role in modulating cell metabolism, differentiation, growth and invasion, we hypothesized that decreased placental LIN28A and/or LIN28B expression impairs trophoblast differentiation and invasion, which could contribute to the development of PE. To address this gap in the knowledge, we evaluated: 1) LIN28 expression in human term placentas and compared LIN28A and LIN28B expression in placentas from gestational-aged matched normotensive with preeclamptic pregnancies,

2) the impact of LIN28 over/under expression on trophoblast invasiveness and differentiation, and 3) the role of hypoxia in regulating LIN28 expression in trophoblasts.

Based on our findings, we propose the hypothesis that deficiencies in LIN28B may be a contributing factor in the initial impaired invasion observed in PE and in the development of the long-term adverse health outcomes associated with the disorder.

Chapter 3

Materials and Methods

Ethical approval

Collection of 15 human placentas from normotensive pregnancies and 13 human placentas from PE affected pregnancies was approved by the Institutional Review Board

(IRB) of the University of South Florida (protocol #00015578) after obtaining informed and written patient consent. Additional total RNA from 6 normotensive and 6 PE pregnancies was provided by Dr. Ananth Karumanchi, Beth Israel Deaconess Medical Center, Harvard

Medical School, Boston, MA. Placentas from the first trimester (7 weeks gestation and 8 weeks gestation, n=2) and early pregnancies (20 weeks gestation, n=2) were obtained from voluntary terminations of uncomplicated pregnancies under the same institutional approval. The clinical characteristics of the patients used for this study are shown in Table

1 and Table 2.

Criteria for inclusion in the study

For the samples used in this study, preeclampsia was diagnosed according to criteria established by The American College of Obstetricians and Gynecologists (194).

According to these guidelines, preeclampsia is defined by blood pressure that is

>140mmHg systolic or >90mmHg diastolic, accompanied by proteinuria, in a woman who was normotensive throughout the first 20 weeks of gestation.

Placental tissue collection

Following delivery, the placenta was placed on ice until dissection. For RNA and protein analysis, tissue samples were excised from the fetal compartment of the placenta, placed in 15 ml conical tubes, and frozen on dry ice. The tissues were then stored at -

80oC until further use. For immunohistochemistry, tissue samples were excised from the fetal compartment of the placenta, fixed overnight in 10% formalin, followed by routine paraffin-embedding procedure. Paraffin blocks were stored at room temperature until further use.

Isolation and culturing of primary DCs

Term DCs were isolated, as previously described from uncomplicated pregnancies undergoing repeat cesarean delivery at term under Yale human investigation committee approval (#22674) (195). Briefly, after removal of the amnion from placental membranes, decidua parietalis was scraped from the chorion, whereas decidua basalis was scraped from the placental surface distal to the intervillous space. The scraped tissues were each digested by collagenase-deoxyribonuclease, purified on a Percoll gradient, grown to confluence, and passaged until found to be >99% free of CD45+ cells by flow cytometry

(195). Confluent term DCs were then primed in serum-containing medium with E2 + MPA

(Sigma-Aldrich, St. Louis, MO) for 7 days to maintain their decidualization properties.

Total RNA was then extracted from confluent DCs and stored at -80oC for use in further studies.

Isolation and culturing of primary CTs

Isolation of CTs from placentas obtained from uncomplicated pregnancies and repeat cesarean deliveries at term using procedures approved by the Yale human investigation committee (#22674) was performed as described (195, 196). Briefly, villous tissue was initially digested with trypsin/deoxyribonuclease I and centrifuged on a discontinuous Percoll gradient (50%/45%/35%/30%). Cells migrating to the 35%/45%

Percoll interface were recovered by centrifugation and were immunopurified using negative selection by simultaneous treatment with mouse anti-human CD9 antibody, mouse anti-human CD45 antibody, and then goat anti-mouse IgG-conjugated

DynaBeads (Life Technologies, Grand Island, NY), yielding CTs with >98% purity on the basis of immunocytochemical and flow cytometric analysis. The decidua basalis layer was trimmed off before digestion, leaving primarily villous tissue for CT isolation. Total

RNA was then extracted and stored at -80oC for further use.

Isolation and culturing of primary STs

STs were obtained by spontaneous differentiation of CTs after 72 hours of culture, as previously described (197). Briefly, CTs were isolated from villous placental tissue at term followed by Percoll gradient centrifugation, as described above. Following isolation,

CTs were grown in DMEM-F12 supplemented with 10% FBS (Gibco Laboratories,

Gaithersburg, MD) for 72 hours to allow for spontaneous syncytialization. Total RNA was then isolated and stored at -80oC for use in later analyses.

Immunohistochemistry

Immunohistochemistry was performed as previously described (198). Briefly, 5-µm paraffin sections from human placentas were incubated overnight at 56oC then deparaffinized in xylenes and rehydrated by graded ethanol washes. Antigen retrieval was performed by boiling in 10 mM citric acid, pH 6.0 for 15 minutes. The sections were then incubated in 3% H2O2 for 12 minutes to quench endogenous peroxidase activity followed by blocking in 5% normal goat serum for 30 minutes at room temperature. The sections were subsequently incubated overnight in a humidified chamber with the primary antibody (rabbit anti-LIN28B, 40μg/ml, Abcam, ab71415 RRID:AB_2135050) diluted in

5% normal goat serum. LIN28B immunoreactivity was detected using a biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, BA-1000 RRID:AB _2313606) together with the avidin-biotin-peroxidase complex (Vectastain ABC kit, Vector

Laboratories, pk6200 RRID:AB_2336826) followed by 3,3’-Diaminobezidine (DAB;

Vector Laboratories, sk-4100 RRID:AB_2336382) incubation as a substrate. The sections were then counterstained with hematoxylin (Vector Laboratories, H3404

RRID:AB_2336843), rehydrated, and covered with a glass coverslip for later analysis.

Quantification of immunohistochemistry

LIN28B immunoreactivity was semi-quantitatively evaluated using the histology score (H-score) method, as previously described (199). Briefly, three different high-power fields for each section were imaged and scored by three separate blinded individuals using the following scale: 0+ (no staining or weak staining), 100+ (moderate and detectable staining), 200+ (distinct staining). The average score for each section was considered the H-score.

Cell culture

BEWO cells (ATCC CCL-98, Manassas, VA, USA) were cultured in F-12K medium supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA).

JEG3 cells (ATCC HTB-36, Manassas, VA, USA) were grown in MEM (GIBCO, Waltham,

MA, USA) supplemented with 10% FBS. HTR-8/SVneo (ATCC CRL-3271, Manassas,

VA, USA) cells were cultured in RPMI-1640 supplemented with 5% FBS. Cell lines were

o maintained at 37 C and 5% CO2.

Cell line authentication

The JEG3, BEWO, and HTR8/SVneo cells were authenticated by short tandem repeat (STR) analysis through ATCC. For authentication, ATCC amplifies 17 short tandem repeats, plus Amelogenin, for comparison to their database of authenticated cell lines. Each of the cell lines used in this study were a 100% match to the STR data in the

ATCC database (appendix 1).

Transfections

JEG3 cells were transfected with scrambled shRNA or an shRNA targeting LIN28B

(OriGene Technologies TG311156, Rockville, MD, USA) using Lipofectamine 2000 following the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA, USA).

HTR8/SVneo cells were transfected with green fluorescent protein (GFP) or pFRT/FLAG/HA-DEST LIN28B plasmid, a gift from Thomas Tuschl (Addgene 43798,

Cambridge, MA, USA) using 25kD linear polyethylenimine (Polysciences 23966,

Philadelphia, PA, USA) (158). Briefly, 105 cells/well were seeded in triplicate in a 12-well plate, plasmids were diluted in sterile water followed by the addition of NaCl (final concentration of 150mM) and polyethylenimine (final concentration of 0.05µg/μl). The mixture was vortexed, incubated at room temperature for 15 minutes and 500µl of RPMI containing 5% FBS medium was added, and the transfection mixture was pipetted onto cells in each well.

Cell proliferation analysis

Cell proliferation was measured using the WST-1 cell proliferation reagent (Sigma

Aldrich, St. Louis, MO, USA), as previously described (200). Briefly, JEG3 or

HTR8/SVneo cells were transfected as indicated above. 72 hours following transfection, the medium was replaced with 90µl fresh medium and 10µl WST-1 reagent. Following incubation at 37oC for 1 hr, the absorbance was measured at 450nm and 690nm, with the latter being used as the reference wavelength. The relative proliferation was then determined for each treatment.

Hypoxia experiments

BEWO or JEG3 cells were seeded in 12-well plates and allowed to attach overnight

o while being housed under normal culture conditions (21% oxygen, 5% CO2) at 37 C in standard CO2 water-jacketed cell culture incubators. The following day, the medium was replaced with fresh medium and the ‘normal culture condition’ (21% oxygen) cells were placed back in the same incubator. The cells that were to be subjected to hypoxic conditions (1% oxygen) were placed in a polycarbonate, airtight hypoxia incubator

41 chamber (StemCell Technologies, Catalog #27310) and flushed with a gas mixture of 1% oxygen, 5% CO2, and 94% nitrogen for 10 minutes at a flow rate of 20L/minute. The sealed chamber was then placed back in the same 37oC water-jacketed incubator. The chamber was flushed at a flow rate of 20L/minute for 10 minutes every 24 hours for the duration of the experiment. Oxygen levels were monitored using a portable oxygen meter

(KKmoon #803) and averaged 1.13 ± 0.14% oxygen throughout the duration of the experiments.

RNA extractions

Placental samples were suspended and homogenized in Qiazol to isolate total

RNA. After experimental cell incubation, cultured cells were lysed using Qiazol. Total RNA was extracted using the miRNeasy mini kit according to the manufacturer’s protocol

(Qiagen).

Protein extractions

For protein isolation, placental specimens were homogenized on ice in lysis buffer containing 20 mM Tris-HCl, 124 mM NaCl, 4 mM EDTA, 0.4% CHAPS, 20 mM HEPES,

200 μM bestatin, 60 μM E-64, 40 μM leupeptin, 600 μM aporotinin, 2 mM NaF, 2.3 mM sodium molybdate, 2 mM sodium orthovanadate, and 1mM b-glycerophosphate. The homogenized samples were then incubated on ice for 20 minutes followed by centrifugation for 10 minutes at 21,000xg. Each resulting supernatant was transferred to a new tube and stored at -80oC for subsequent immunoblotting. For protein isolation from cell culture experiments, collected cells were pelleted then resuspended in lysis buffer

42 and incubated on ice for 20 minutes, followed by centrifugation for 10 minutes at

21,000xg. The supernatant was then stored at -80oC for later immunoblotting.

Immunoblotting

Protein lysates were fractionated by SDS-PAGE as previously described (201).

Following fractionization, the lysates were transferred to a nitrocellulose membrane and subsequently blocked for 1 hour at room temperature in Odyssey blocking buffer (LI-

COR). The membranes were then incubated overnight with LIN28B (2μg/ml, Abcam, ab71415 RRID:AB_2135050) and α-tubulin (0.35μg/ml, Cell Signaling Technology,

2144S RRID:AB_2210548) primary antibodies followed by probing with the secondary antibody IRDye 680 goat anti-rabbit (0.2μg/ml, LI-COR, 926-68073 RRID:AB_10954442).

The resulting immunoblots were scanned and quantitatively assessed using the Odyssey infrared imaging system (LI-COR).

qRT-PCR

cDNA was synthesized by reverse transcription of 500 ng of total RNA using M-

MuLV reverse transcriptase per the manufacturer’s protocol (New England Biolabs,

M0253L). After reverse transcription, relative mRNA expression levels were measured by using TaqMan Fast Advanced Mastermix (Life Technologies, 4366072) and TaqMan qPCR primers/probes (ThermoFisher Scientific, LIN28A: HS_00702808_s1, LIN28B:

HS_01013729_m1, TNFα: HS_01113624_g1, SYN-1: HS_01926764_u1, ITGβ4:

HS_00236216_m1, ELABELA: HS_04274421_m1) with the Quantstudio 3 qPCR machine (Applied Biosystems). For miRNA expression analysis, 50ng of total RNA was

43 reverse transcribed using a TaqMan miRNA reverse transcription kit (Applied Biosystems,

4366597) and miRNA specific TaqMan primers/probe sets (ThermoFisher Scientific, miR-

516a: 002416, miR-516b: 001150, miR-517a: 002402, miR-518c: 002401, miR-519d:

002403, Let-7g: 478580, U18: 001204) as previously described (202). The experimental results were then analyzed using the delta-delta Ct method.

Scratch wound assays

HTR8/SVneo cells were seeded in a 24-well dish and transfected the following day with a plasmid encoding either GFP or LIN28B. 72 hours later, as previously described, a single scratch wound was established in each well using a P-200 pipette tip (203). The well was then washed thoroughly with sterile PBS. The wounds were then photographed to establish the baseline for later comparison. After 24 hours incubation in the full medium, the cultured cells were fixed in PFA and stained with crystal violet and re-photographed for analysis of wound closure.

Trans-well migration assays

Trans-well migration assays were performed as previously described (204, 205).

Briefly, HTR8/SVneo cells were seeded in a 12 well dish and transfected the following day with a plasmid encoding GFP (control) or LIN28B. After 72 hours, 2x104 transfected cells were seeded in serum free RPMI in the upper chamber of a Matrigel coated trans- well insert (insert: Falcon, 353097, Matrigel: Corning 354744). RPMI containing 5% FBS was placed in the lower chamber. 24 hours later, non-migrated cells were removed from the upper chamber using a cotton tipped swab. The remaining migrated cells were fixed

44 with PFA and then stained with crystal violet and imaged. The number of migrated cells/field was counted to determine the migration.

Statistical analyses

All results are presented as means ± SEM. All data were tested for normality and equivalence of variance to determine the appropriate statistical tests. One-way ANOVA with Tukey’s post hoc test was performed for multiple group analyses. Multiple two-way

Students t-tests with Holm-Sidak correction for multiple comparisons were performed to determine changes in mean gene expression. For analysis of demographic data,

Students t-tests, chi-square test, or Fisher’s exact test were used to determine significant differences in the cohorts. All statistical analyses were performed using GraphPad Prism

7 software and results were considered significant when adjusted p<0.05.

Table 1. Available demographic data and clinical characteristics of the 15 normal and 13 PE subjects obtained from the University of South Florida for this study.

Characteristic Normal n=15 Preeclampsia n=13 Maternal Median age 25 [19-36] 26 [19-33] Gravida 2 [1-12] 2 [1-4] Median Gestational Age 39 [37-40.1] 37.6 [37.0-40.3] BMI 32.8 [24-62.6] 31.3 [29-77.2] Maternal Race White 3 [20%] 6 [46.2%] Black 5 [33.3%] 3 [23.1%] Asian 1 [6.7%] 0 [0%] Hispanic 4 [26.7%] 4 [30.8%] Unknown 2 [13.3%] 0 [0%] Cigarette Use 2 [13.3%] 2 [15.4%] cHTN 1 [6.7%] 2 [15.4%] DM (Gestational DM) 4 (2) [26.7%] 2 (1) [15.4%] Mode of Delivery Vaginal 4 [26.7%] 4 [30.8%] Cesarean section 11 [73.3%] 9 [69.2%] Indication for C/S Failure to Progress 0 [0%] 4 [30.8%] * Other 0 [0%] 1 [7.7%] Repeat 9 [60%] 2 [15.4%] Fetal Sex Male 4 [26.7%] 8 [61.5%] Female 11 [73.3%] 5 [38.5%] Fetal Weight (g) 3390 [2590-4440] 3380 [1965-3960] *Indicates p<0.05 vs normal cohort

Table 2. Available demographic data and clinical characteristics of the 6 normal and 6 PE subjects obtained from the Harvard Medical School for this study.

Characteristic Normal n=6 Preeclampsia n=6 Maternal Median age 35 [23-39] 25 [23-37] * Gravida 2.6 [1-6] 1.5 [1-4] Median Gestational Age 39 [39-40] 34 [30-39] * BMI 24.1 [19.5-29.3] 26.9 [19.6-35.4] Maternal Race White 6 [100%] 4 [66.7%] Asian 0 [0%] 2 [33.3%] Cigarette Use 0 [0%] 1 [16.7%] cHTN 0 [0%] 1 [16.7%] DM (Gestational DM) 0 (0) [0%] 0 (0) [0%] Mode of Delivery Vaginal 0 [0%] 2 [33.3%] Elective C/S 6 [100%] 4 [66.7%] Indication for C/S Failure to Progress 0 [0%] 0 [0%] Other 3 [50.0%] 2 [33.3%] Repeat 3 [50.0%] 2 [33.3%] Fetal Sex Male 2 [33.3%] 5 [83.3%] Female 4 [66.7%] 1 [16.7%] Fetal Weight (g) 3917 [3240-4420] 2217 [1045-3905] * *Indicates p<0.05 vs normal cohort

Chapter 4

Decreased LIN28B in Preeclampsia Impairs Human Trophoblast Differentiation

and Migration

Introduction

PE is a life-threatening complication of pregnancy with clinical symptoms that include hypertension and proteinuria, and is a leading cause of maternal morbidity worldwide (1). In severe cases, PE can result in tonic-clonic seizures and/or hemolysis, elevated liver enzymes, and low platelet counts, conditions known as eclampsia and

HELLP syndrome, respectively (206). Currently, the comprehensive underlying etiology of PE is incompletely understood. However, it appears that PE is generally driven due to a maternal milieu that is susceptible to hypertension and/or chronic exposure of the placenta and developing fetus to prolonged pathologically hypoxic conditions (7, 8, 207).

In early human pregnancy, invasive EVTs begin invading the decidual tissue following implantation of the blastocyst to the uterine wall. Through several different mechanisms, this invasion ultimately results in extensive remodeling of the maternal spiral arteries leading to myogenic tone loss and adequate perfusion of the placenta (69,

77, 208). However, in PE this EVT invasion is impaired, which results in inadequate spiral vasculature remodeling and insufficient perfusion of the placenta (97, 98). The resulting prolonged hypoxia can result in IUGR and can stimulate the secretion of placentally derived factors, such as sFLT-1 and sEng, into the maternal circulation, ultimately resulting in systemic endothelial dysfunction and elevation of the maternal blood pressure

(104, 105, 209). Furthermore, ST formation and function is known to be impaired by hypoxia and is often observed in the placentas of PE affected pregnancies (210, 211).

The most extensively characterized function of the RNA binding protein LIN28 is its inhibition of the maturation of the Let-7 family of miRNAs. However, LIN28 is also post- transcriptionally regulated by the Let-7 family. This highly conserved LIN28/Let-7 switch plays an integral role in regulation stem cell self-renewal, cellular differentiation, embryonic growth, developmental timing, cellular glucose metabolism, and cellular invasion (140, 162, 163, 191). In vertebrates, LIN28 exists as two paralogs that share approximately 65% sequence identity, with LIN28B being 41 amino acids larger and containing a C-terminal nuclear localization signal (145). While LIN28A and LIN28B are very similar and share many of the same functions, it is becoming more clear that they also have separate functions in the cell. Furthermore, the role of LIN28 in cellular growth, invasion, metabolism, and differentiation suggests that it may play a key role EVT differentiation and invasion, however their role in the human placenta has yet to be investigated. Furthermore, based on these known cellular roles, aberrant expression of

LIN28 may contribute to the pathogenesis of PE. Therefore, we aimed to evaluate: 1)

LIN28A and LIN28B expression in human term placentas to determine if there is differential expression between the two paralogs, 2) LIN28 expression in placentas from gestational-aged matched normotensive with preeclamptic pregnancies, 3) the impact of

LIN28 over/under expression on trophoblast invasiveness and differentiation, and 3) the role of hypoxia in regulating LIN28 expression in trophoblasts.

Results

LIN28B is the predominant LIN28 paralog expressed in human term placentas

Considering LIN28A and LIN28B may have differing functions within the cell, we first set out to determine if either paralog was preferentially expressed in the human term placenta. To accomplish this, we isolated total RNA from whole placenta tissue lysates and performed qRT-PCR for LIN28A and LIN28B. This analysis revealed that LIN28B was expressed ~1300-fold higher than LIN28A, indicating that LIN28B was the predominant paralog expressed in the human term placenta (Figure 5A). To confirm and expand on these findings, we isolated total RNA from primary cultures of human DCs,

CTs, and STs collected from normal term placentas and performed qRT-PCR analysis for LIN28A and LIN28B. This analysis revealed that LIN28B was expressed ~50, ~750, and ~500-fold higher than LIN28A in DCs, CTs, and STs respectively, further confirming that LIN28B is the predominantly expressed LIN28 paralog in the human term placenta

(Figure 5B).

Figure 5. LIN28B is the predominant LIN28 paralog expressed in human placentas.

A. qRT-PCR analysis for LIN28A and LIN28B mRNA expression normalized to GAPDH in term whole placental tissue lysate. B. qRT-PCR analysis for LIN28A and LIN28B mRNA expression normalized to GAPDH in isolated primary human DCs, CTs and STs. Results represent means ± SEM. * indicates p<0.05 versus LIN28A

LIN28B is expressed significantly higher in CTs and STs compared to DCs in the human term placenta

To determine the precise in situ protein localization of LIN28B in the human term placenta, we performed immunohistochemistry for LIN28B on human term placental specimens and quantified the expression using an H-score method (199). These immunostainings and subsequent quantifications revealed significantly higher levels of

LIN28B in CTs and STs compared to DCs (Figure 6A-D). Collectively, these findings suggested that LIN28B is the predominant paralog expressed in human placentas and its expression is primarily in placental versus decidual cell types. In light of these findings, we primarily focused on the function, expression, and regulation of LIN28B for the remainder of the study.

In situ LIN28B protein is decreased in placentas from PE complicated pregnancies.

Given the predominant expression of LIN28B over LIN28A in the human term placenta, as well as its major role in cellular proliferation, invasion, and metabolism, we next wanted to determine if its expression is reduced in the placentas of PE complicated pregnancies. To accomplish this, we performed immunohistochemical staining for

LIN28B on term placentas from normal and PE complicated pregnancies. Interestingly, as shown in Figure 7A-D, LIN28B expression appears greatly reduced in both invasive trophoblasts and trophoblasts of the chorionic villi of placental specimens from PE affected pregnancies compared to normal pregnancies.

Figure 6. LIN28B is expressed significantly higher in CTs and STs compared to DCs in the human term placenta

A-C. Representative immunohistochemistry images in term human placenta of invasive trophoblasts (A) and villous trophoblasts (B) immunostained for LIN28B or non-immune rabbit IgG only control (C). D. H-score analysis for LIN28B protein expression in DCs, CTs, and STs of placental specimens from normotensive pregnancies. Scale bar represents 35 μm. n= 10 placental sections in (D). Results represent means ± SEM. *indicates p<0.05 versus DCs.

Figure 7. in situ LIN28B protein is decreased in placentas from PE pregnancies.

A-D. Representative images of LIN28B immunostaining of invasive trophoblasts (A and C) and villous trophoblasts (B and D) in placentas from normotensive pregnancies (A and B) and in PE placental tissues (C and D). Scale bar represents 35 μm.

LIN28B mRNA and protein expression is reduced in the placenta of PE complicated pregnancies

To confirm the immunohistochemical findings that suggested LIN28B is reduced in the placenta of PE complicated pregnancies, we isolated total RNA and total protein from normal and PE affected pregnancies. Subsequent qRT-PCR and immunoblot analyses for LIN28B confirmed that both the mRNA and protein expression level was significantly reduced in placental tissue from PE complicated pregnancies compared to placental tissue from normal pregnancies (Figure 8A-C).

To investigate whether there is an increase in LIN28A expression to compensate for the decrease in LIN28B observed in the placenta of PE affected pregnancies, we also performed immunoblot and qRT-PCR analyses for LIN28A protein and mRNA expression on the same samples used for analysis of LIN28B expression. These analyses revealed no significant change in the already low protein or mRNA expression of LIN28A in placental tissue from PE affected pregnancies compared to placenta tissue from normal pregnancies (Figure 9A-C). Collectively, these results confirm that both the mRNA and protein expression of LIN28B, but not LIN28A, is significantly reduced in placental tissue of PE complicated pregnancies, which warrants further investigation into the role of

LIN28B in pregnancy and PE.

Figure 8. LIN28B protein and mRNA is decreased in the placenta of PE complicated pregnancies

A. Immunoblot analysis for α-Tubulin and LIN28B from placenta tissue lysates from four term normotensive pregnancies and four PE-affected pregnancies. B. Densitometric immunoblot analysis for LIN28B normalized to α-tubulin from tissue lysates from 15 normotensive pregnancies and 13 PE-affected pregnancies. C. qRT-PCR analysis for LIN28B mRNA expression normalized to GAPDH in placental lysates from 21 normotensive pregnancies and 19 PE-affected pregnancies. Results represent means ± SEM. * indicates p<0.05 versus normal.

Figure 9. LIN28A protein and mRNA expression is unchanged in the placentas of PE complicated pregnancies

A. Immunoblot analysis for α-Tubulin and LIN28A from placenta tissue lysates from four term normotensive pregnancies and four PE-affected pregnancies. B. Densitometric immunoblot analysis for LIN28A normalized to α-tubulin in term placental tissue lysates from 15 normal pregnancies and 13 PE-affected pregnancies. C. qRT-PCR analysis for LIN28A normalized to GAPDH in term placental tissue lysates from 15 normal pregnancies and 13 PE-affected pregnancies. Results represent means ± SEM.

The Let-7/LIN28B ratio is increased in the placenta of PE complicated pregnancies

The inhibition of Let-7 miRNA maturation by LIN28B has been well documented and characterized (145). Therefore, we aimed to test whether the decrease in LIN28B expression in the placenta of PE affected pregnancies results in increased Let-7 miRNA expression. To accomplish this, we performed qRT-PCR analysis for Let-7g, which is a

Let-7 family member that has been shown to be highly sensitive to LIN28B expression, as well as Let-7a, another member of the Let-7 family of miRNAs (158). As shown in

Figure 10, the Let-7g/LIN28B and Let7-a/LIN28B ratio trended towards an increase

(p=0.072 for Let-7g/LIN28B, p=0.077 for Let-7a/LIN28B) in placental tissues from PE complicated pregnancies.

LIN28B is highly expressed in syncytial sprouts and invasive EVTs in first trimester human placentas

Following implantation, human trophoblasts can differentiate into EVTs, which invade and remodel the maternal spiral arteries, or STs, which form the syncytial layer of the placenta. The population of EVTs includes proliferative proximal EVTs, invasive distal

EVTs, interstitial EVTs in the interstitial area of the decidua, and endovascular EVTs in the maternal spiral arteries, all of which can be detected in first trimester and early pregnancy placental sections. Therefore, we performed immunohistochemical staining for

LIN28B on first trimester and early pregnancy human placental sections to determine the precise in situ LIN28B expression pattern among the different trophoblast types. This analysis revealed that syncytial sprouts and EVTs in the trophoblastic cell columns in anchoring villi displayed stronger LIN28B expression when compared to villous

58 cytotrophoblasts (Figure 11A). Furthermore, LIN28B immunoreactivity gradually increased from the proximal (proliferative) EVTs to the distal (invasive) EVTs in the cell columns (Figure 11B). Moreover, the strongest LIN28B expression was exhibited in interstitial trophoblasts (cytokeratin positive) invading the decidua (vimentin positive)

(Figure 11 C and D). This pattern of LIN28B expression provides supporting in situ evidence for its involvement in the differentiation of trophoblasts towards an invasive phenotype and/or syncytialization.

LIN28B increases trophoblast migration and invasion in vitro

To complement the in situ immunohistochemistry findings that LIN28B is highly expressed in invasive EVTs invading the maternal decidua with functional in vitro experiments, we overexpressed LIN28B in a first trimester human trophoblast cell line

(HTR8/SVneo) and tested its effect on cell migration and cell invasion, using an established scratch wound assay and Matrigel invasion assay, respectively (203-205). As shown in Figure 12, overexpression of LIN28B resulted in a significant increase in

HTR8/SVneo migration when compared to control. Furthermore, overexpression of

LIN28B resulted in an increase in cell invasion through the Matrigel, however it did not reach statistical significance (p=0.053, Figure 13). Collectively, these analyses indicate that LIN28B plays a major role the in regulation of trophoblast migration and invasion, both in vitro and in human tissue.

Figure 10. The Let-7/LIN28B ratio trends towards an increase in the placenta of PE complicated pregnancies

A. Let-7g/LIN28B protein ratio determined by qRT-PCR analysis for let-7g normalized to U18 and LIN28B immunoblot normalized to α-tubulin in placental lysates from normotensive pregnancies and PE-affected pregnancies. B. Let-7a/LIN28B protein ratio determined by qRT-PCR analysis for let-7a normalized to U18 and LIN28B immunoblot normalized to α-tubulin in placental lysates from normotensive pregnancies and PE- affected pregnancies. Results represent means ± SEM.

Figure 11. LIN28B is highly expressed in syncytial sprouts and invasive trophoblasts in first trimester placentas A-C. Representative images of immunohistochemical staining for LIN28B with nuclear hematoxylin counterstain in human first trimester placental sections. LIN28B is highly expressed in the syncytial sprouts of chorionic villi (black arrows, A). LIN28B expression gradually increases from proliferative proximal EVTs (PEVTs) to distal invasive EVTs (DEVTs) in placental cell columns (B). LIN28B expression is lowest in PEVTs and increased in invasive interstitial trophoblasts (black arrows) invading the maternal decidua (red arrowheads) in anchoring villi (C). D. Representative serial section of tissue shown in (C) double immunostained for decidual cell marker vimentin (pink) and trophoblast marker cytokeratin-7 (brown). Red arrowheads and black arrows in (D) correspond to red arrowheads and black arrows in (C). Scale bar represents 35µm.

Figure 12. LIN28B increases HTR8/SVneo cell migration

A. Top: representative images at the time of scratch wound application of HTR8/SVneo cells transfected with GFP (control) or LIN28B. A. Bottom: representative images of cells stained with crystal violet 24 hours after scratch wound application. B. Quantification of migration 24 hours after scratch wound application. Results represent means ± SEM. *indicates p<0.05 versus control.

Figure 13. LIN28B increases HTR8/SVneo cell invasion

A. Representative crystal violet staining image of HTR8/SVneo cells that migrated through Matrigel 72 hours after transient transfection with GFP (control) or LIN28B. B. Quantification of invaded cells from (A). Five high power fields were imaged per insert for analysis. Results represent means ± SEM.

Decreased LIN28B expression reduces trophoblast proliferation To determine whether the reduced LIN28B expression observed in the placentas of PE complicated pregnancies also affects trophoblast proliferation, LIN28B was knocked down in JEG3 cells or overexpressed in HTR8/SVneo cells (Figure 14A and B).

These cell lines were chosen because the human trophoblast-like cell line JEG3 cells has high basal LIN28B expression, while HTR8/SVneo cells, a human first trimester trophoblast cell line, has undetectable levels of LIN28B expression. As shown in Figure

15, transient knockdown of LIN28B in JEG3 resulted in a ~35% decrease in cell proliferation, whereas overexpression of LIN28B in HTR8/SVneo cells resulted in a ~40% increase in cell proliferation. Taken together, these data indicate that LIN28B expression not only regulates trophoblast migration and invasion, but also proliferation.

LIN28B expression regulates trophoblast differentiation

Considering the well-established role of LIN28B in the regulation of cell differentiation, along with our data indicating LIN28B is highly expressed in invasive

EVTs, STs and syncytial sprouts (Figure 6 and 11), we next sought to determine whether

LIN28B regulates the differentiation of trophoblasts in vitro. As shown in Figure 16A, transient knockdown of LIN28B in JEG3 cells resulted in a ~29% reduction of the mRNA level of the ST differentiation marker SYN-1. Furthermore, this transient knockdown of

LIN28B in JEG3 cells resulted in a ~50% increase in the mRNA expression of ITGβ4, an integrin subunit enriched on non-invasive villous trophoblasts (Figure 16A). We also performed qRT-PCR analysis for SYN-1 and ITGβ4 in HTR8/SVneo cells following transient overexpression of LIN28B, however there was no detectable significant change in the expression of these genes in this cell line (Figure 16B). Taken together, this

64 invasion, migration, proliferation, and differentiation data indicates that LIN28B expression regulates trophoblast differentiation towards a syncytial or invasive phenotype.

LIN28B expression regulates inflammation

Aberrant expression of inflammatory cytokines, in both the mother and placenta, is a characteristic of PE. In particular, TNFα has been shown to be increased in both the maternal circulation and the placenta of pregnancies complicated by PE (130, 131).

Therefore, we aimed to determine if the decreased LIN28B expression levels in the placenta of PE complicated pregnancies could potentially result in differential expression of TNFα. To answer this question, we performed qRT-PCR for TNFα mRNA expression following transient knockdown or overexpression of LIN28B in JEG3 and HTR8/SVneo cells, respectively. As shown in Figure 17A, transient knockdown of LIN28B in JEG3 cells results in a ~50% increase in TNFα mRNA expression. Furthermore, transient overexpression of LIN28B in HTR8/SVneo cells results in a ~42% decrease in the mRNA expression of TNFα (Figure 17B). Taken together, these experiments provide in vitro evidence that LIN28B regulates the expression of TNFα.

Figure 14. Transient knockdown of LIN28B in JEG3 cells and transient overexpression of LIN28B in HTR8/SVneo cells A. Immunoblot (top), densitometric quantification (left) and qRT-PCR analysis (right) for LIN28B expression in JEG3 cells 72 hours after transient transfection with scrambled shRNA (control) or and shRNA targeting LIN28B. B. Immunoblot (top), densitometric quantification (left) and qRT-PCR analysis (right) for LIN28B expression in HTR8/SVneo cells 72 hours after transient transfection with GFP (control) or plasmid encoding LIN28B. Results represent means ± SEM. *p<0.05 versus scrambled. #p<0.05 versus control.

Figure 15. LIN28B expression regulates trophoblast proliferation A. Cell proliferation analysis of JEG3 cells 72 hours after transient transfection with scrambled shRNA or an shRNA targeting LIN28B. B. Cell proliferation analysis of HTR8/SVneo cells 72 hours after transient transfection with GFP (control) or plasmid encoding LIN28B. Results represent means ± SEM. *indicates p<0.05 versus scrambled. #indicates p<0.05 versus control.

Figure 16. LIN28B expression regulates trophoblast differentiation A. qRT-PCR analysis for SYN-1 and ITGβ4 in JEG3 cells 72 hours after transient transfection with a scrambled shRNA or shRNA targeting LIN28B. B. qRT-PCR analysis for SYN-1 and ITGβ4 in HTR8/SVneo cells 72 hours after transient transfection with GFP (control) or plasmid encoding LIN28B. Results represent means ± SEM. *indicates p<0.05 for shLIN28B versus scrambled.

Figure 17. LIN28B expression regulates TNFα expression A. TNFα expression in JEG3 cells 72 hours following transient transfection of scrambled shRNA or shRNA targeting LIN28B. B. TNFα expression in HTR8/SVneo cells 72 hours following transient transfection with GFP (control) or plasmid encoding LIN28B. Results represent means ± SEM. *indicates p<0.05 versus scrambled. #indicates p<0.05 versus control.

Decreased LIN28B expression reduces the expression of ELABELA

ELABELA is a placentally derived circulating peptide hormone and is an endogenous ligand for the apelin receptor. It has recently been shown that mice deficient in ELABELA exhibit PE-like symptoms, such as elevate blood pressure and proteinuria, likely due to impaired placental angiogenesis (212). Considering that LIN28B is an RNA binding protein that has been shown to bind and regulate the expression of thousands of genes, we aimed to determine if reduced LIN28B expression in PE affected ELABELA expression. Interestingly, as shown in Figure 18A, transient knockdown of LIN28B resulted in a ~40% reduction in ELABELA mRNA expression. To complement these findings, we measured ELABELA mRNA expression in HTR8/SVneo cell following transient overexpression of LIN28B, however these cells do not express ELABELA and overexpression of LIN28B did not induce its expression (Figure 18B).

LIN28B regulates the placenta and ESC specific chromosome 19 miRNA cluster

The C19MC is a large miRNA cluster that is exclusively expressed in the placenta, in ESCs, and in some tumors. Furthermore, miRNAs of the C19MC have been reported to be decreased in the placenta of PE complicated pregnancies (126). Considering

LIN28B is an RNA binding protein that plays a role in the regulation of miRNA expression and is also almost exclusively expressed in the placenta, ESCs, and some cancers, while also decreased in PE (Figure 7 and 8), we aimed to determine if reduced LIN28B expression resulted in decreased C19MC expression. To make this determination, we performed qRT-PCR analyses for 5 randomly chosen miRNAs encoded throughout the length of the C19MC following transient knockdown of LIN28B. As shown in Figure 19,

70 knockdown of LIN28B resulted in a ~30-50% decrease in all five miRNAs, three of which

(miR-516a, miR-516b, and miR-519d) reached statistical significance.

Hypoxia decreases LIN28B expression in BEWO and JEG3 cells

Shallow implantation in PE is associated with inadequate EVT invasion and impaired remodeling of the spiral arteries, thus resulting in insufficient placental perfusion and progressively hypoxic conditions (97, 213). Thus far, the results presented here indicate that trophoblasts are the main cell type expressing LIN28B, and that LIN28B plays a major role in trophoblast migration, invasion, proliferation, and differentiation.

Therefore, we assessed the effect of hypoxia on LIN28B expression in trophoblasts. To accomplish this, JEG3 cells were cultured for 72 hours in hypoxia (1% O2), which resulted in a >50% reduction in the mRNA and protein expression levels of LIN28B when compared to standard culture conditions (21% O2) (Figure 20). To complement this experiment, we also subjected BEWO cells, another trophoblast cell line with high basal levels of LIN28B (Figure 21A), to 72 hours of hypoxia and found that the LIN28B mRNA and protein expression levels were significantly reduced (Figure 21A-C). These results indicate that LIN28B expression is regulated by hypoxia, which may have implications in

PE.

Figure 18. Decreased LIN28B expression reduces ELABELA expression A. qRT-PCR analysis for ELABELA in JEG3 cells 72 hours after transient transfection with scrambled shRNA or shRNA targeting LIN28B. B. qRT-PCR analysis for ELABELA in HTR8/SVneo cells 72 hours after transient transfection with GFP (control) or LIN28B. ELABELA expression was undetectable in both conditions in HTR8/SVneo cells. Results represent means ± SEM. *indicates p<0.05 versus scrambled.

Figure 19. Decreased LIN28B results in reduced expression of the C19MC qRT-PCR analysis for the indicated miRNA of the C19MC normalized to U18 in JEG3 cells 72 hours after transient transfection with scrambled shRNA or shRNA targeting LIN28B. Results represent means ± SEM. *indicates p<0.05 for shLIN28B versus scrambled

Figure 20. Hypoxia reduces LIN28B protein and mRNA expression in JEG3 cells A-C. Immunoblot analysis for α-tubulin and LIN28B expression (A), normalized densitometric quantification of LIN28B protein expression (B), and qRT-PCR analysis for LIN28B mRNA expression (C) in JEG3 cells following culture for 72 hours in standard culture conditions (21% oxygen) or hypoxia (1% oxygen). Results represent means ± SEM. *indicates p<0.05 for 1% oxygen versus 21% oxygen.

Figure 21. Hypoxia reduces LIN28B protein and mRNA expression in BEWO cells A-C. Immunoblot analysis for α-tubulin and LIN28B expression (A), normalized densitometric quantification of LIN28B protein expression (B), and qRT-PCR analysis for LIN28B mRNA expression (C) in BEWO cells following culture for 72 hours in standard culture conditions (21% oxygen) or hypoxia (1% oxygen). Results represent means ± SEM. *indicates p<0.05 for 1% oxygen versus 21% oxygen.

Hypoxia decreases LIN28A expression in JEG3 and BEWO cells

To determine if LIN28A is subject to the same hypoxic regulation as LIN28B, we cultured JEG3 cells in standard culture conditions (21% oxygen) or hypoxia (1% oxygen) for 72 hours and measure the LIN28A protein and mRNA expression. As shown in Figure

22, LIN28A protein and mRNA were significantly reduced following culturing in hypoxia.

Upon performing this same experiment in BEWO cells, we again found that LIN28A protein and mRNA is significantly reduced by hypoxia (Figure 23). These results indicate that LIN28A is regulated by hypoxia in a manner similar to that of LIN28B.

Hypoxia increases Let-7a and Let-7g expression in JEG3 and BEWO cells

To test if there was a concomitant increase in Let-7 miRNA expression with the reduction in LIN28A and LIN28B, we performed qRT-PCR for Let-7a and Let-7g in JEG3 and BEWO cells following 72 hours of culture in hypoxia. As shown in Figure 24, we found that expression of both Let-7a and Let-7g were increased, although this increase did not attain statistical significance.

Hypoxia decreases SYN-1 and ELABELA and increases TNFα in JEG3 and BEWO cells

Considering knockdown of LIN28B led to reduced SYN-1 and ELABELA mRNA expression levels, while increasing TNFα mRNA expression levels, and culture in hypoxia led to significantly reduced LIN28B expression levels, we aimed to test the effect of hypoxia on these markers of trophoblast differentiation and inflammation. Similar to

LIN28B knockdown experiments, culturing of BEWO and JEG3 cells for 72 hours in hypoxic conditions resulted in a >60% (p<0.05) reduction the mRNA levels of SYN-1, and

>1000% increase (p<0.05) increase in expression of TNFα in both cell lines compared to standard culture conditions (Figure 25). Furthermore, culturing JEG3 cells in hypoxic conditions reduced the expression of ELABELA to undetectable levels, while exposure of

BEWO cells to hypoxic condition resulted in a ~30% decrease in ELABELA expression, but did not reach statistical significance (Figure 25). However, unlike LIN28B knockdown experiments, hypoxic conditions had no effect on the mRNA expression of ITGβ4.

Figure 22. Hypoxia reduces LIN28A protein and mRNA expression in JEG3 cells A-C. Immunoblot analysis for α-tubulin and LIN28A expression (A), normalized densitometric quantification of LIN28A protein expression (B), and qRT-PCR analysis for LIN28A mRNA expression (C) in JEG3 cells following culture for 72 hours in standard culture conditions (21% oxygen) or hypoxia (1% oxygen). Results represent means ± SEM. *indicates p<0.05 for 1% oxygen versus 21% oxygen.

Figure 23. Hypoxia reduces LIN28A protein and mRNA expression in BEWO cells A-C. Immunoblot analysis for α-tubulin and LIN28A expression (A), normalized densitometric quantification of LIN28A protein expression (B), and qRT-PCR analysis for LIN28A mRNA expression (C) in BEWO cells following culture for 72 hours in standard culture conditions (21% oxygen) or hypoxia (1% oxygen). Results represent means ± SEM. *indicates p<0.05 for 1% oxygen versus 21% oxygen.

Figure 24. Hypoxia increases Let-7a and Let-7g in JEG3 and BEWO cells A and B. qRT-PCR analysis for Let-7a and Let-7g in JEG3 cells (A) and BEWO cells (B) after culture for 72 hours in standard culture conditions (21% oxygen) or hypoxia (1% oxygen. Results represent means ± SEM.

Figure 25. Hypoxia reduces SYN-1 and ELABELA and increases TNFα in trophoblasts A and B. qRT-PCR analysis of the indicated gene in JEG3 cells (A) and BEWO cells (B) after 72 hours of culture in standard culture conditions (21% oxygen) or hypoxic conditions (1% oxygen). Results represent means ± SEM. *indicates p<0.05 for 1% oxygen versus 21% oxygen standard culture conditions.

Discussion

Many cases of PE appear to arise from insufficient EVT invasion accompanied by impaired remodeling of the maternal spiral arteries resulting in insufficient blood flow to the developing fetal-placental unit. This impaired EVT invasion results in extended periods of pathologically hypoxic conditions for the growing placenta and fetus. Ultimately, this local placental hypoxia is a likely contributor to, or marker of, long-term adverse effects on maternal health as well as fetal growth restriction caused by placental insufficiency (214, 215). The mechanisms responsible for the development of PE and its longstanding adverse health effects, for both the mother and child after birth, are complex and under active investigation. Results from the current study revealed that the RNA binding protein, LIN28B, is expressed at significantly higher levels than LIN28A in the human placenta and is significantly down-regulated at both the mRNA and protein levels in placental tissues of term pregnancies affected by PE. Furthermore, the let-7g/LIN28B ratio is increased in placentas from PE-complicated compared to normotensive pregnancies. The current study also demonstrated that increased LIN28B expression is associated with increased EVT invasiveness, both in the placenta in situ and in vitro as displayed by immunohistochemistry, scratch wound assays, and Matrigel invasion assays. Knockdown of LIN28B in JEG3 cells resulted in a significant decrease in SYN-1,

ELABELA and miRNAs of the C19MC cluster, and an increase in expression of the non- invasive trophoblast marker ITGβ4, as well as the pro-inflammatory cytokine TNFα.

Furthermore, exposure to hypoxia resulted in decreased LIN28A and LIN28B expression in the trophoblast-like cell lines BEWO and JEG3. Lastly, exposure to hypoxia decreased

SYN-1 and ELABELA mRNA expression, while it increased TNFα and Let-7 expression in BEWO and JEG3 cells.

This is the first study demonstrating that LIN28B is the predominant paralog of

LIN28 expressed in the human placenta. LIN28B expression is confirmed to be higher than LIN28A in isolated primary DCs, CTs, and STs. Moreover, in situ LIN28B protein is expressed significantly higher in CTs and STs when compared to DCs. LIN28B and

LIN28A share 65% sequence identity and have similar structures that include N-terminal cold shock domain, C-terminal Zinc-knuckle domain, and putative nucleolar localization sequences. Although, LIN28B has 41 more amino acids and contains C-terminal domain with a nuclear localization signal, although both can function in the nucleus and cytosol

(216). While it is known that LIN28A and LIN28B share similar functions, such as regulation of let-7 maturation and regulation of thousands of mRNAs, the predominant expression of LIN28B in human placental tissue suggests that LIN28B has a unique role in human placenta. For instance, stable isotope labeling by amino acids in cell culture

(SILAC) studies in stable LIN28B knockdown cell lines have shown that loss of LIN28B results in changes in protein expression of thousands of genes, many of which regulate progression through the cell cycle (217). Therefore, it is likely that, in addition to regulating the expression of Let-7, LIN28B plays a major role in regulating the expression of many other genes and the cell cycle in placental cells during development.

This study also shows that LIN28B expression is decreased in human placentas from PE affected pregnancies. We suspect that this decrease in LIN28B is being driven, at least in part, by placental hypoxia. However, more studies are needed to determine the precise upstream factors leading to the reduction in LIN28B observed in this study. Global

83 decreases in LIN28B expression, on both the mRNA and protein level, were observed which indicates changes in transcription and/or post-translational changes resulting in protein degradation. Accompanying the decrease in LIN28B observed in this study is an increase in the Let-7g and Let-7a miRNA/LIN28B protein ratio. This increase is in agreement with previous studies that have shown that LIN28B selectively binds to a common GGAG motif in the loop region of pri-Let-7 and pre-Let-7 transcripts, resulting in either inhibition of DROSHA (pri-Let-7) or DICER1 RNase III (pre-Let-7) processing (154,

155, 218-220). In humans, the Let-7 miRNA family contains twelve members and the suppression of Let-7g, Let-7b, and Let-7f by LIN28B has been well documented, although

Let-7b and Let-7f were not tested in this study (158, 221). Based on the well-characterized and strong inhibition of maturation of Let-7g by LIN28B, it is no surprise that Let-7g levels appear increased in placentas in which LIN28B levels are decreased. Furthermore,

LIN28B has been shown to inhibit the maturation of other miRNAs, such as miR-107, miR-143, and miR-200c, although the expression of these miRNAs in the placenta was not tested in this study (222, 223). Potential changes in the expression of these miRNAs due to decreased LIN28B expression are important to keep in mind when considering changes in gene expression associated with PE.

The disruption in the LIN28B/Let-7 pathway observed in this study may have major implications in long-term health of the fetus. For instance, the LIN28B/let-7 switch has been shown to be a major contributor in the control of cellular metabolism through the regulation of genes in the insulin-PI3K-mTOR pathway (162). Expression of LIN28A and

LIN28B has been shown to increase the activating phosphorylation of Akt at serine 473, thus suggesting activation of the PI3K-mTOR pathway. Furthermore, this activating

84 phosphorylation is abrogated by the overexpression of Let-7, providing further evidence that the LIN28/Let-7 switch plays a major role in cellular metabolism. Elegant in vivo studies have also implicated the LIN28/Let-7 switch in the involvement of glucose metabolism and homeostasis. For instance, embryonic knockout of LIN28B in mice leads to stunted postnatal growth and greatly impaired glucose tolerance, whereas mice engineered to overexpress LIN28B grow much larger than wild-type littermates, exhibit increased sensitivity to insulin, and display higher glucose metabolism (162, 163).

Furthermore, fetal muscle-specific LIN28B knockout resulted in decreased adult growth, and long-term adverse health effects that mimic metabolic disorder, such as insulin resistance, and impaired glucose uptake compared to wild-type controls, which suggests a crucial role for pre-natal LIN28B expression in both pre-natal and post-natal development (180). On the other hand, mice engineered to overexpress let-7 displayed similar phenotypes to LIN28B knockout mice. For instance, they were smaller than wild- type littermates, had impaired glucose tolerance whether fed a normal or high fat diet, and displayed insufficient insulin secretion (162, 164). These results, again, lend more evidence to suggest that pre-natal LIN28B expression plays a major role in long-term health outcomes. In addition to transgenic and knockout mouse studies, human GWAS studies have linked LIN28B to multiple long-term outcomes, including adult height, age of both puberty and menarche and the development of type 2 diabetes (224-227). Given all of this mounting evidence, it is becoming more clear that early LIN28B expression is a major factor involved in long-term health and development. Therefore, the reduced

LIN28B expression in the placentas of PE-complicated pregnancies observed in this study may contribute to the explanation of why children born from PE-complicated

85 pregnancies are often born smaller for gestational age compared to children from unaffected pregnancies and have a much greater risk of developing long-term detrimental health outcomes, such as metabolic and cardiovascular related diseases including diabetes and heart disease (23, 228).

Proper initiation and progression of pregnancy requires sufficient implantation and

EVT invasion into the decidua followed by extensive remodeling of the maternal spiral vasculature. The EVT invasion and remodeling during early pregnancy is strikingly similar to that of cancer metastasis, with the two processes sharing several similar molecular pathways (181, 229). In agreement with this, LIN28B has been reported to increase cancer cell proliferation and invasion, and its expression has been correlated with poor prognosis of multiple cancers, such as hepatocellular carcinoma, acute myeloid leukemia, brain cancer, breast cancer, and many others (141, 183, 187). The development of PE is frequently associated with impaired EVT invasion accompanied by poor remodeling of the spiral arteries resulting in decreased blood flow required by the developing fetal- placental unit and prolonged pathological hypoxia throughout pregnancy. Accordingly, the results of this study demonstrate that invasive interstitial EVTs invading the maternal decidua in 1st trimester human placental sections express higher levels of LIN28B compared to non-invasive proximal EVTs. Furthermore, overexpression of LIN28B increases proliferation, migration and invasion of first trimester HTR8/SVneo cells in vitro.

Conversely, this study is the first to show that knockdown of LIN28B in JEG3 cells decreases cellular proliferation and led to an increase in ITGβ4, an integrin subunit that is highly expressed on the surface of non-invasive trophoblasts and helps to regulate cell- cell adhesion and cell adhesion to the extracellular matrix. Taken together, these findings

86 suggest that high expression levels of LIN28B are required for proper trophoblast proliferation and invasion into the maternal decidua. It is possible that LIN28B is contributing to this invasion through stimulation of EMT, a process which trophoblasts undergo during the transition from non-invasive villous CTs to invasive EVTs, and which

LIN28B has been shown to induce (190, 192). It is also possible that high LIN28B expression is contributing to trophoblast invasiveness indirectly through the repression of

Let-7, which results in increased expression of extracellular matrix remodeling factors and/or growth factors. For instance, Hmga1 and Hmga2 are known targets of Let-7, and their expression has been shown to upregulate MMPs, resulting in increased capacity for extracellular matrix remodeling (230-234). Therefore, the high level of LIN28B expression and the resulting low level of Let-7 expression could potentially be an underlying mechanism of increased MMP secretion and trophoblast invasion. However, future studies are needed to identify the precise molecular pathway with which LIN28B contributes to trophoblast migration and invasion into the decidua.

Although LIN28B is generally known to inhibit the maturation of miRNAs, this study has shown that knockdown of LIN28B in JEG3 cells results in reduced the expression of several miRNAs of the C19MC cluster. While it is not currently known if LIN28B directly binds any of these miRNAs, these surprising results could potentially be explained by epigenetic changes. For instance, the CpG-rich region upstream of the C19MC is prone to hypermethylation, which results in reduced C19MC expression, while demethylation of the region increases C19MC expression (235). Furthermore, using CHIP-seq analysis a recent study showed that, in addition to its RNA binding capabilities, LIN28 can bind directly to specific consensus sequences in DNA promoter regions (236). Binding of

LIN28A to these specific regions leads to the recruitment of the CpG demethylase TET1, thus demethylating the regions and activating transcription, while knockdown of LIN28A results in global decreases in the expression of many genes due to decreased demethylation (236). Therefore, it is possible that the knockdown of LIN28B in JEG3 cells shown herein results in increased methylation of CpG-rich regions upstream of C19MC, and thus reduced expression. Moreover, further analysis of recently published PAR-CLIP data indicates that LIN28B can interact with CpG rich-Alu repeats that are dispersed throughout the C19MC cluster (158). In fact, these CpG rich-Alu repeats are some of the most abundant genomic elements encoded throughout the C19MC. Importantly, these

CpG rich-Alu repeats can function as independent promoters for RNA polymerase III, thus transcriptionally activating and driving expression of the miRNAs of the C19MC (237-

239). Furthermore, we recently showed that overexpression of LIN28B in HEK293 cells, a cell line which does not typically express LIN28B or the C19MC, resulted in expression of some of the miRNAs of the C19MC (240). Considered together, our data showing that knockdown of LIN28B results in decreased C19MC expression, while overexpression of

LIN28B leads to increased C19MC expression, strongly implies that C19MC expression is, at least in part, dependent on LIN28B expression. Therefore, the decreased LIN28B expression in the placentas of PE-complicated pregnancies described herein may contribute to the altered expression of C19MC reported in many cases of PE (126).

However, further studies are needed to elucidate the specific pathways through which suppression of LIN28B results in decreased C19MC expression and whether other changes in gene expression are due to a reduction in these miRNAs.

We report here for the first time that knockdown of LIN28B in JEG3 cells resulted in a significant reduction in the mRNA level of SYN-1. However, overexpression of

LIN28B in HTR8/SVneo cells did not result in increased expression of SYN-1, which indicates that LIN28B alone is likely not sufficient to induce its expression. SYN-1 is known to induce cellular membrane fusion and is highly expressed in STs of the placenta.

Furthermore, SYN-1 has been shown to be decreased in the placenta of PE complicated pregnancies, contributing to the overall impaired syncytialization (100, 241). In addition to the reduced SYN-1 expression observed upon knockdown of LIN28B, we also show that LIN28B is very highly expression in the syncytium of term placentas and the syncytial sprouts of first trimester placentas, further indicating a potential link between LIN28B,

SYN-1, and syncytialization. Together, these knockdown and immunohistochemical experiments provide the first evidence that decreased LIN28B expression in PE could potentially be a contributing factor to the decreased SYN-1 expression and impaired syncytialization observed in the placentas of PE complicated pregnancies. However, the direct mechanism underlying these gene expression changes as yet to be elucidated.

SYN-1 mRNA is not a predicted target of the Let-7 family of miRNAs or other miRNAs known to be inhibited by LIN28B, such as miR-107, miR-143, and miR-200c. Therefore, the changes in SYN-1 expression associated with changes in LIN28B expression are likely due to indirect mechanisms that are not yet characterized.

Several pro-inflammatory cytokines are documented to play a role in endothelial dysfunction and endothelial activation, with aberrations in their expression levels reported in the development of PE. One pro-inflammatory cytokine, TNFα, has been reported to be elevated in both the amniotic fluid and the maternal plasma of pregnancies affected

89 by PE. Considering LIN28B has been shown to interact with thousands of mRNA transcripts to promote or repress their translation, we tested whether LIN28B expression levels affect TNFα expression and found that knockdown of LIN28B results in elevated levels of TNFα mRNA. Conversely, we show that overexpression of LIN28B in

HTR8/SVneo cells results in significantly reduced TNFα mRNA expression. This study provides the first evidence that LIN28B expression regulates TNFα expression and identifies a potential link between decreased LIN28B expression in the placenta and the pro-inflammatory conditions presented in PE. Analysis of previously published RNA pull down data indicates that LIN28B does not directly interact with TNFα mRNA, thus the mechanism behind the increase in TNFα upon knockdown of LIN28B is likely indirectly due to decreased LIN28B expression (158). For instance, human TargetScan analysis reveals that the C19MC miRNAs miR-516b, miR-519, and miR-525 are predicted to target the 3’UTR of TNFα. Therefore, the increased TNFα expression observed upon knockdown of LIN28B could potentially be due to decreased repression by the C19MC, considering C19MC miRNA expression is reduced when LIN28B is knocked down.

However, future studies are warranted to determine the specific link between LIN28B and the complex inflammatory signaling in PE in general and in regulating TNFα in particular.

ELABELA is a peptide hormone that is highly expressed in the developing embryo, placenta, serum, and some adult tissues. As an endogenous ligand for the apelin receptor, ELABELA has been shown to regulate vascular tone, angiogenesis, and vascular development (242). In addition to its proangiogenic functions, it has been demonstrated that treatment of trophoblasts with ELABELA results in increased trophoblast invasiveness in vitro (212). Furthermore, mice deficient in ELABELA, but not

90 the apelin receptor, display symptoms of PE such as proteinuria and elevated blood pressure, with exogenous delivery of ELABELA reversing the PE-like symptoms (75). In this study, we show for the first time that knockdown of LIN28B in JEG3 cells results in decreased expression of ELABELA. However, we also show that HTR8/SVneo cells do not express ELABELA, and its expression is not induced following overexpression of

LIN28B. This lack of expression in HTR8/SVneo cells could be due to a lack of necessary transcription factor expression or hypermethylation of the ELABELA promoter region. It has been previously shown that LIN28B can bind thousands of mRNA transcripts, leading to increased translation and increased gene expression. However, a previous study using an RNA pull down assay has shown that LIN28 does not bind to ELABELA mRNA, therefore the changes in ELABELA expression in JEG3 cells due to knockdown of LIN28B observed herein must be through an indirect mechanism, rather than direct interaction of

LIN28B and ELABELA (243). ELABELA is not a predicted target of the Let-7 family of miRNAs or other miRNAs known to be inhibited by LIN28B, therefore future studies are needed to identify the underlying mechanism responsible for decreased ELABELA expression following LIN28B knockdown.

ITG4 is an integrin subunit that is enriched on the surface of non-invasive, villous trophoblasts and decreased on the surface of extravillous invasive trophoblasts. In this study we show that knockdown of LIN28B in JEG3 cells results in an increase in the expression of ITG4, indicating that decreased LIN28B expression results in a less invasive trophoblast phenotype. However, overexpression of LIN28B in HTR8/SVneo cells resulted in only a moderate decrease in ITG4 expression, suggesting that LIN28B expression alone is not enough to alter the integrin expression pattern in this cell type.

While the exact mechanism underlying the increased ITG4 expression upon knockdown of LIN28B in JEG3 cells has not been elucidated, it could be potentially explained by post- transcriptional miRNA regulation. For instance, human TargetScan analysis reveals that the C19MC miRNAs miR-524 and miR-519 are predicted to target the 3’UTR of ITGβ4.

Therefore, it is plausible that the reduced C19MC expression following knockdown of

LIN28B shown herein could result in increased ITGβ4 expression due to decreased repression of the mRNA transcript. However, further studies are necessary to tease out the specific mechanisms underlying these changes.

It is established that insufficient placental perfusion results in increasingly pathological utero-placental hypoxia. The experiments shown herein are the first to report that exposure of BEWO or JEG3 cells to hypoxia results in a decrease in the expression of LIN28A and LIN28B mRNA and protein, with an accompanying increase in Let-7 miRNA expression. It has recently been shown that LIN28A can be post-translationally modified by MAPK/ERK, with this phosphorylation resulting in increased protein stability

(244). Furthermore, it has also been demonstrated that MAPK/ERK is downregulated by hypoxia in ESCs, while hypoxia has been shown to induce the expression of Let-7 in endothelial and cancer cell lines (245-247). Thus, this decrease in stabilizing phosphorylation, or the increase in Let-7 expression, could conceivably lead to the decreased LIN28A and LIN28B expression following hypoxia reported in this study. This study is the first of its kind to show that LIN28B is reduced by hypoxia, which has major implications for stem cells, embryogenesis, and/or development, all of which can be impacted by hypoxia. The exact mechanism underlying this reduction, as well as its long- term implications warrants further investigation.

In addition to the decrease in LIN28A and LIN28B and the increase in Let-7, exposure of JEG3 and BEWO cells to hypoxia lead to reduced mRNA expression of SYN-

1 and ELABELA. Based on human TargetScan analysis neither SYN-1 nor ELABELA are predicted to be targets of Let-7, thus their reduced expression is likely due to another mechanism (248). Considering SYN-1 and ELABELA are also both significantly reduced upon knockdown of LIN28B, it is plausible that LIN28B directly or indirectly regulates the expression of these genes, as it is an RNA binding protein that can target many different mRNA transcripts. Collectively, the LIN28B- and hypoxia-dependent reduction in SYN-1 and ELABELA expression likely directly relates to placental insufficiency due to impaired angiogenesis and a reduction in syncytiotrophoblast formation, which play an integral role in maternal-placental gas and nutrient transport (249, 250). However, future studies aiming to elucidate the specific mechanism behind these reductions are warranted.

It is plausible that very early on in pregnancy, well before the manifestation of measurable clinical symptoms, a currently unknown factor causes LIN28B expression to be reduced in placentas of PE affected pregnancies. Based on the well-established roles of LIN28B regarding pluripotency, EMT, and cellular proliferation and invasion, in addition to the data and observations shown in this study, decreased LIN28B expression during blastocyst development, specifically trophectoderm development, could conceivably result in impaired implantation and insufficient trophoblast invasion and remodeling of the maternal spiral vasculature. Thus, this impaired spiral artery remodeling would result in prolonged pathological hypoxia thereby further decreasing LIN28B expression. This detrimental feed-forward loop could be an underlying factor in the development of PE

(Figure 26). However, it is also possible that placental hypoxia triggered by an unknown

93 mechanism leads to the downregulation of LIN28B in PE. Future studies are required to elucidate the specific mechanism behind the reduction in LIN28B expression observed herein.

As explained by the development origin of health and disease (DOHaD) hypothesis, environmental factors such as poor nutrition of the mother or fetus, among many others, interfere with normal fetal development and likely contribute to long-term adverse health outcomes, such as detrimental changes in the metabolism of the child

(251). However, the exact signal transduction pathways and cellular mechanisms underlying this phenomenon are not currently known. Considering fetal LIN28B expression is highly correlated with development and long-term changes in growth and metabolism, it is pertinent to investigate whether the decreased LIN28B expression in placentas from PE-complicated pregnancies contributes to the adverse adulthood health outcomes (162, 163, 180). Therefore, we propose the hypothesis that reduced LIN28B expression may be a contributing underlying factor in the DOHaD since it is known that:

1) LIN28B plays a major role in the regulation of long-term cellular growth and metabolism; 2) fetal knockout of LIN28B results in long-term adverse health conditions such as reduced growth and impaired glucose metabolism; and 3) LIN28B is reduced in

PE, which is associated with long-term adverse changes in metabolism. Therefore, it is plausible that decreased LIN28B expression during development is an underlying factor in these changes. Future studies directed at testing this hypothesis may confirm that

LIN28B contributes to the central mechanism of the DOHaD and may lay the foundation for future epidemiological and molecular studies.

Figure 26. Schematic model of the contribution of LIN28B to the normal physiology of pregnancy and the pathological conditions of PE

A. Simplified model of normal pregnancy in which there exists high levels of LIN28B, leading to sufficient trophoblast invasion, adequate spiral artery remodeling, and proper placental perfusion. B. Simplified schematic model showing the contribution of decreased LIN28B to the development of PE and PE-associated metabolic disorders.

Chapter 5

Limitations and Overall Conclusions

Limitations

One limitation to the studies performed herein is the use of the trophoblast-like choriocarcinoma cells (JEG3 and BEWO) and immortalized cells derived from first trimester villous explants (HTR8/SVneo). Although the use of these cells, which are generally accepted for use as in vitro trophoblast models, make performing gene manipulation studies easier, the use of primary derived human trophoblasts would add more translational relevance. Unfortunately, due to culturing constraints of primary isolated human trophoblasts, the experiments performed would not be feasible. Upon isolation and culture from a freshly delivered placenta, primary trophoblasts begin to spontaneously differentiate into STs after only 2 to 3 days. For the experiments performed in this study, cells transfected with an shRNA targeting LIN28B or a plasmid encoding

LIN28B were allowed to grow for 3 days following transfection to permit sufficient knockdown of the endogenously expressed protein or exogenous protein overexpression.

For these experiments to be performed in primary isolated human trophoblasts, the cells would have to be isolated, seeded at the proper density, then transfected and allowed to grow for 3 days to permit gene knockdown/overexpression. The numerous gene and phenotypic changes that occur during the spontaneous differentiation into syncytiotrophoblasts could confound the outcome of the experiments. Therefore, we chose to use the cells lines depicted herein for these experiments. In addition to the use

96 of choriocarcinoma derived trophoblast-like cells, another potential cell culture limitation in this study is the use of 21% oxygen (i.e. normal culture conditions) for comparison to the culture in hypoxia (1% oxygen). Physiologically, 21% oxygen would be considered hyperoxia, considering all tissues in the body are exposed to blood-oxygen concentrations significantly lower than this. Comparison of LIN28B expression levels in trophoblasts cultured in hypoxia (1% oxygen), as well as other more physiologically accurate oxygen concentrations, such as 3%, 6%, and 10%, would add additional value to these studies.

Another potential limitation to this study is differences in the clinical data of the normal and PE affected cohorts. For instance, there was a marked difference in the indication for cesarean section (CS) between the two cohorts. Specifically, 13 individuals in the PE cohort underwent CS, 4 of which were due to failure to progress, while 16 individuals in the normal cohort underwent CS, with the majority being repeat CS and none due to failure to progress. It is plausible that this discrepancy could influence the findings of this study. However, we identified a widespread decrease in the expression of

LIN28B, on both the mRNA and protein level. These observed changes are suggestive of changes in the stability and degradation of LIN28B protein, as well as changes in transcription. Therefore, these differences in LIN28B expression are not likely due to the relatively short time between induction of labor and performance of a CS due to the failure to progress. However, further studies using more samples from normal and PE affected pregnancies that undergo CS due to failure to progress could be conducted to address whether the failure to progress and performance of a CS affect the post-delivery placental expression of LIN28B.

A third potential limitation to the study is the fact that we measured LIN28B expression in term placentas of normal and PE affected pregnancies. Therefore, we cannot conclusively say that decreased LIN28B causes PE; we can only speculate that decreased LIN28B is associated with and contributes to the pathophysiology of PE. It would be more informative to measure the expression level of LIN28B early on in pregnancy, when the underlying pathophysiological changes associated with PE are taking place, to determine if reduced LIN28B expression results in decreased spiral artery remodeling and ultimately development of PE. Unfortunately, these experiments are not feasible due to only having access to early pregnancy placentas that are from pregnancies that have been terminated. We currently have no way of knowing whether

PE would have been developed during the pregnancy. This limitation could potentially be avoided by the use of animal models of PE. However, current rodent models of PE fail to fully recapitulate the human condition, and the placental implantation and development process in rodents is quite different than that of humans.

Overall conclusions

To our knowledge, this study is the first of its kind to examine the expression and function of the RNA binding proteins LIN28A and LIN28B in the context of the human placenta and pregnancy. Despite the large amount of literature characterizing the function of LIN28A and LIN28B, prior to these studies it was not known which paralog, if either, was preferentially expressed in the human placenta. Furthermore, the well characterized role of LIN28A and LIN28B in regulating cellular proliferation, growth, metabolism, differentiation, invasion, and migration suggests that LIN28A or LIN28B may play a role

98 in the invasion of EVTs into the maternal decidua. Therefore, we hypothesized that decreased placental LIN28A and/or LIN28B expression impairs trophoblast differentiation and invasion, which could contribute to the development of PE.

In order to address the role and function of LIN28A or LIN28B in the human placenta, we first determined if either paralog is preferentially expressed. Using whole placental tissue lysates from normal pregnancies, we found that LIN28B is expressed over 1000-fold higher than LIN28A. Furthermore, we found that LIN28B is expressed significantly higher than LIN28A in primary isolated human DCs, STs, and CTs, with the expression being primarily in placental STs and CTs, rather than maternal DCs.

Moreover, in situ protein localization further confirmed that LIN28B expression is primarily in the STs and CTs, as opposed to the DCs. Moving forward, we next tested whether

LIN28B is decreased in the placentas of PE complicated pregnancies and found that it is significantly reduced on both the mRNA and protein levels, and there is no compensatory increase in LIN28A expression. To test if this decreased LIN28B expression may be an underlying contributing factor to the development of PE, we examined the LIN28B expression in first trimester placentas, when EVT invasion into the maternal decidua is prevalent. Using differential immunohistochemical staining, we found that LIN28B is highly expressed in syncytial sprouts and its expression increases from proximal, non- invasive, to distal invasive EVTs, with LIN28B expression being the highest in EVTs invading the maternal decidua and very little LIN28B expression in the decidual cells.

These findings confirm that LIN28B is reduced in PE, and that it plays a role in early EVT invasion into the maternal decidua. To support these findings with functional in vitro tests, we overexpressed LIN28B and found that this led to increased cell migration, invasion,

99 and proliferation. Furthermore, knockdown of LIN28B expression resulted in decreased cellular proliferation.

Since LIN28B is an RNA binding protein that is known to target thousands of genes, we next aimed to test whether LIN28B expression regulates inflammation or trophoblast differentiation, as well as other markers associated with PE. Upon knockdown of LIN28B in JEG3 cells, we observed a decrease in the syncytial marker SYN-1, accompanied by an increase in the villous trophoblast maker ITGβ4, suggesting that

LIN28B plays a role in the differentiation of trophoblasts to an invasive and/or syncytial phenotype. In addition, knockdown of LIN28B resulted in an increase in the proinflammatory cytokine TNFα, which is also increased in PE, while overexpression of

LIN28B reduced TNFα expression. Moreover, LIN28B knockdown led to reduced expression of miRNAs of the C19MC, as well as reduced expression of ELABELA, a circulating peptide hormone associated with PE. Collectively, these findings provide evidence for the involvement of LIN28B in the differentiation of human trophoblasts.

Since the development of PE is associated impaired EVT invasion and exposure of the placenta and fetus to prolonged pathological conditions, we tested the effect of hypoxia on LIN28B. These experiments showed that LIN28B is drastically reduced upon exposure to hypoxia, and this reduction in LIN28B is associated with decreased SYN-1 expression and increased TNFα expression; results that mirror the effect of shRNA mediated LIN28B knockdown. In total, these results show that LIN28B is reduced upon exposure to hypoxia, and this reduction is associated with increased inflammation and changes in trophoblast differentiation, which may be important in the pathology of PE.

100

PE is associated with many short- and long-term adverse health outcomes for both the mother and child. For instance, children born from PE complicated pregnancies are often born early and/or born small for gestational age. Furthermore, adults who were exposed to PE in utero have a significantly elevated risk of developing cardiovascular disorders, such as hypertension, and metabolic disorders, such as diabetes, when compared to adults who were born from normotensive pregnancies. However, the underlying mechanism to these long-term adverse health outcomes is not currently known. Recently, LIN28B has been shown to play a major role in the regulation of cellular growth and metabolism. For instance, fetal knockout of LIN28B in mice results in mice that are born smaller than wild type littermates. Furthermore, these mice are prone to obesity, and develop glucose intolerance and insulin resistance. Moreover, mice engineered to overexpress LIN28B have increased lean weight, improved glucose tolerance and insulin sensitivity, and are resistant to the development of obesity when fed a high fat diet. Collectively, these elegant mouse studies indicate that fetal LIN28B expression plays a major role in long-term health and well-being. Considering loss of fetal

LIN28B expression results in long-term impaired glucose metabolism and that PE is often associated with long-term adverse health outcomes, coupled with our data that shows

LIN28B is reduced in PE, we propose the hypothesis that decreased LIN28B expression in PE may be an underlying contributing factor for the development of the adverse health outcomes associated with PE. Future studies aimed at testing this hypothesis may confirm LIN28B as an underlying factor in the developmental origins of health and diseases.

101

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Appendix A

JEG3 ATCC cell line authentication STR analysis profile report

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HTR8/SVneo ATCC cell line authentication STR analysis profile report

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BEWO cell line authentication STR analysis profile report

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USF IRB approval for collection of human placental specimens

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