MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation Of Minqian Shen Candidate for the Degree Doctor of Philosophy _______________________________ Dr. Haifei Shi, Advisor _______________________________ Dr. Kathleen Killian, Committee Chair _______________________________ Dr. Jack Vaughn, Committee _______________________________ Dr. James Janik, Committee _______________________________ Dr. Xiaowen Cheng, Graduate School Representative ABSTRACT ROLES OF ESTROGEN HORMONES AND ESTROGEN RECEPTORS ON REGULATION OF LIVER AND LIVER CANCER METABOLISM by Minqian Shen Liver is one of the most essential organs involved in the regulation of energy homeostasis. Hepatic steatosis, a major manifestation of metabolic syndrome, is associated with imbalance between lipid formation and breakdown and glucose production and catabolism. Although the most common risk factors of hepatocellular carcinoma (HCC) are hepatic virus infection and alcohol, a rapid increase in obesity has become a prime cause of HCC, outweighing HCC with virus- or alcohol- related etiology. Estrogens affect both cell metabolism and proliferation. Both nuclear estrogen receptors (ERs), ER-α and ER-β, are expressed in the liver. How estrogen hormones regulate liver homeostasis and which receptors play the fundamental role in the metabolic process are not clear. Moreover, whether estrogen hormones and ERs are protective or detrimental in HCC is under debate, and whether estrogens can alter HCC metabolism and interfere with leptin-induced HCC is not known. The goal of my dissertation is to further elucidate the roles of estrogens and ERs in normal liver metabolism as well as HCC metabolism and proliferation using both in vivo and in vitro models. Thus, I hypothesize that 1) exogenous estrogen replacement reverses liver metabolic alteration in estrogen-depleted mice mainly through ER-α receptor; 2) estrogens inhibit leptin-induced HepG2 cell proliferation mainly through ER-β activation and alter HepG2 metabolism mainly through ER-α activation. In this dissertation, previous studies on the functions of estrogen and estrogen receptors in liver and liver cancer are discussed in Chapter 1. Studies using ovariectomized mouse model with hormone-replacement was applied to determine the role of estrogens and estrogen receptors in mouse liver metabolism are discussed in Chapter 2. Human cancer cell line HepG2 was treated with different concentrations of estrogen and estrogen receptor agonists as well as estrogen receptor siRNA to determine the role of estrogen and estrogen receptors on HepG2 cancer cell proliferation, apoptosis and leptin signal pathway in Chapter 3. High performance liquid chromatography (HPLC) was used to determine the role of estrogen and estrogen receptors on HepG2 cancer cell gene expression and metabolic profiles in Chapter 4. In Chapter Five, I conclude the finding of my current studies and give perspectives for future research directions of estrogen hormones and estrogen receptors in the regulation of liver and liver cancer metabolism. A DISSERTATION Presented to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology by Minqian Shen The Graduate School Miami University Oxford, Ohio 2017 Dissertation Director: Dr. Haifei Shi TABLE OF CONTENTS TABLE OF CONTENTS…………………………………………………………………………iii LIST OF TABLES……………………………………………………………………………..….v LIST OF FIGURES……………………………………………………………..………………..vi LIST OF ABBREVIATIONS………………………………………………………………..….viii ACKNOWLEDGEMENTS……………………………………………………………………....x Chapter 1. Introduction of estrogens’ role in liver metabolism and hepatocellular carcinoma 1.1 Obesity and metabolic syndrome ……………………………………………………..…..…1 1.2 Physiologic function of estrogens in liver…………………………………………......……..2 1.3 Glucose and lipid metabolism in liver……………………………………………...……...…4 1.4 Link between obesity and hepatocellular carcinoma…………………………………………5 1.5 Estrogen’s effects on hepatocellular carcinoma………………………………………………6 1.6 Summary…………………………………………………………………………………...…7 References…………………………………………………………………………...……………9 Figure legends……………………………………………………………………………………14 Chapter 2. Estrogens and estrogen receptors regulate energy metabolism in liver in mouse model Abstract…………...…………………………………………………………...…………………16 2.1 Introduction…………………..………………………………………………………………17 2.2 Methods and materials……………………………………………………………………….19 2.3 Results…………………………………………………………………………………….….23 2.4 Discussion.…….………………………………………………………………………….….29 References………………………………………………………………………………………..32 Figure legends……………………………………………………………………………..……..35 Chapter 3. Estrogen and estrogen receptors on HepG2 cancer cell proliferation, apoptosis and leptin signal pathway. iii Abstract…………………………………………………………………………………………..53 3.1 Introduction………………………………………………………………………………….54 3.2 Methods and materials……………………………………………………………………….55 3.3 Results………………………………………………………………………………………..58 3.4 Discussion……………………………………………………………………………....……60 References…………………………………………………………………………..……………65 Figure legends……………………………………………………………………………………71 Chapter 4. Estrogen and estrogen receptors on HepG2 cancer cell metabolic profile Abstract…………………………………………………………………………………………..84 4.1 Introduction……………………………………………………………………………….….85 4.2 Methods and materials……………………………………………………………………….86 4.3 Results………………………………………………………………………………………..90 4.4 Discussion……………………………………………………………………………………93 References…………………..……………………………………………………………………95 Figure legends……………………………………………………………………………………97 Chapter 5. Conclusion and perspectives………………………………………………………..119 References………………………………………………………………………………………122 iv LIST OF TABLES Table 2.1 Primers for qPCR……….…………………………………………………………….38 Table 4.1 Top biological functions identified by Ingenuity Pathway Analysis regulated by E2, PPT and DPN…………………………………………………………………………………….99 Table 4.2 qPCR primers for RNA sequencing check…………………………………………...107 Table 4.3 qPCR primers for metabolic enzymes……………………………………………….108 v LIST OF FIGURES Figure 1.1 Genomic effects of estrogens and androgens via nuclear ERs and ARs……………..12 Figure 1.2 Metabolic effects of estrogens and androgens on regulation of lipid, glucose, and cholesterol in the liver…………..………………………………………………………………..12 Figure 2.1 Body weight, food intake and circulating E2………………………...………………39 Figure 2.2 Body composition, peri-gonadal fat weight and circulating leptin…………………..40 Figure 2.3 Plasma lipid profiles………………………………………………………………….41 Figure 2.4 Liver TG, liver weight, liver/whole body weight ratio and ADP/ATP ratio……….…42 Figure 2.5 Insulin sensitivity test and glucose tolerance test…………………….………………43 Figure 2.6 Insulin sensitivity signal pathway……………………………………………………44 Figure 2.7 Gene expression levels involved in energy metabolism………………………….…..45 Figure 2.8 Metabolic profiles………………………………………………………………….…47 Figure 3.1 Effects of leptin, 17β-estradiol, and estrogen receptor agonists on HepG2 cell number in cell culture……………………………………………………………………..…………..…..74 Figure 3.2 Effects of leptin, 17β-estradiol, and estrogen receptor agonists on proliferation of HepG2 cells………………………………………………………………………………………75 Figure 3.3 Effects of leptin, 17β-estradiol, and estrogen receptor agonists on apoptosis of HepG2 cells…………………………………….………………………………………………………...76 Figure 3.4 Effects of leptin on estrogen receptor expression in HepG2 cells……………………77 Figure 3.5 Effects of leptin, 17β-estradiol, and estrogen receptor agonists on STAT3 and SOCS3 signaling in HepG2 cells…………………………………………………………………………78 Figure 3.6 Effects of leptin, 17β-estradiol, and estrogen receptor agonists on ERK and p38/MAPK signaling in HepG2 cells……………………………………………………………80 Figure 3.7 Effects of ER siRNAs on cell proliferation and activation of leptin signaling………82 Figure 4.1 Effects of sodium oxamate (OX), oligomycin (OM), 2-deoxy-D-glucose (2-DG), and 17β-estradiol (E2) on HepG2 cell number in cell culture…………………………..…………..109 Figure 4.2 Effects of different doses of sodium oxamate (OX), oligomycin (OM), 2-deoxy-D- vi glucose (2-DG) on HepG2 cytotoxicity, viability and apoptosis……………………………….110 Figure 4.3 Effects of E2, PPT and DPN on gene expression levels involved in energy metabolism……………………………………………………………………………………...111 Figure 4.4 RNA sequencing data shows different gene expression upon E2, PPT and DPN treatments on HepG2 cells……………………………………………………………………...113 Figure 4.5 Evaluation of target genes identified by RNA sequencing…………………………114 Figure 4.6 Metabolic profiles…………………………………………………………………..115 vii LIST OF ABBREVIATIONS E2 17β-Estradiol DPN 2,3-bis(4-hydroxy-phenyl)-propionitrile 2-DG 2-deoxy-D-glucose PPT 4,4’,4’’-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol 6Pfk 6-phosphofructokinase Acc acetyl co-enzyme carboxylase ArKO aromatase-knockout BMI body mass index ChREBP carbohydrate regulated element-binding protein Cox6a cytochrome c oxidase ER estrogen receptor ER-α estrogen receptor α ER-β estrogen receptor β ERE estrogen response element FA fatty acid FASN fatty acid synthase FFA free fatty acid GTT glucose tolerance tests GLUT glucose transporter Glut2 glucose transporter 2 Gapdh glyceraldehyde-3-phosphate dehydrogenase Pygl glycogen phosphrylase Gys2 glycogen synthase 2 GPER G-protein coupled estrogen receptor HCC hepatocellular carcinoma HDL-C high-density lipoprotein cholesterol HFD high-fat diet HPLC–MS high-performance liquid chromatography-mass spectrometry IR insulin receptor ITT insulin tolerance test IL-6 Interleukin-6 ip intraperitoneal LPL lipoprotein lipase viii LDL-C