The Interaction between Intestinal Cells and Bacteria
The Effect of Butyrate on Gene Expression
Brigita Meškauskaité, Göcke Nihan Yildirim, Marion Kračmerová and Mariam Labrouzi Supervised by: Jesper Troelsen
Roskilde University Fourth Semester, Fall 2015 International Bachelor in Natural Sciences, House 14, NSM
ABSTRACT
More and more research is being done on the symbiotic relation between bacteria and the human body. Bacteria reside in various places - one of them being the intestine - where they contribute with various important molecules such as sodium butyrate that is used as energy in colonic epithelial cells. The overall aim of the project was to establish whether there is an interaction between bacteria residing in the intestinal lumen and the intestinal epithelial cells, by investigating the effect of butyrate on specific genes. The method used is a promoter analysis by transfection of reporter plasmids into Caco-2 cells. The results are obtained by measuring the activity of the reporter genes.
According to the results obtained from our procedure, we were able to see that sodium butyrate had an affect on the expression of several genes. TCF4, APC, HBP1, SOX9, CGN, CDCX2, CDC42, CDKN2D, YAP-1, HNF4, SPINT-1, Oca2 and HOXB4 were upregulated while TOPFlash, Heph and miR194a were downregulated. Further, it was established, based on the genes analyzed, that butyrate might be related to the NF-KB complex and Wnt signaling pathway. However, these findings cannot be considered as conclusive, more extensive research needs to be done in order to fully understand the mechanisms that are affected by sodium butyrate and how they induce the up or downregulation of specific genes. Further research within this area could allow the discovery of more important factors involved in the interaction between bacteria and human cells.
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TABLE OF CONTENTS
ABSTRACT ...... 2 TABLE OF CONTENTS ...... 3 AIM ...... 4 INTRODUCTION...... 5 THE INTESTINE ...... 6 Epithelial Cells ...... 6 Bacteria in the Intestine...... 9 Sodium Butyrate ...... 10 GENE REGULATION ...... 12 Gene Transcription ...... 12 Regulation of Gene Expression ...... 13 Gene Regulation in the Intestine ...... 14 NF-ΚB protein complex ...... 15 Wnt signaling pathway ...... 17 THEORY BEHIND THE METHODS...... 19 Caco-2 cells - a model of colon cancer cells ...... 19 Experimental Analysis of Gene Expression ...... 20 METHODS ...... 25 RESULTS ...... 28 Optimization ...... 28 Butyrate experiment ...... 31 DISCUSSION ...... 40 CONCLUSION ...... 47 PERSPECTIVES ...... 48 REFERENCES ...... 49 APPENDIX ...... 55
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AIM
The main aim of this project is to examine the interaction between bacteria that reside in the intestine and the human epithelial cells. Sodium butyrate, a product secreted by bacteria in the intestinal lumen, is selected to represent this interaction.
Scientific aim: Examine butyrate’s effect on the human gene expression. Technical aim: Set up an artificial system that will represent the influence of butyrate on the gene expression.
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INTRODUCTION
Bacteria are usually considered harmful for the human organism as they can cause serious infections. There is ongoing research in the field of microbiology, trying to invent new methods of treating bacterial infections. However, there is a symbiotic relationship between millions of bacteria and most parts of the human body that most people are not aware of. Bacteria in the intestine, for example, are very beneficial for the digestion, absorption of nutrients and secretion of essential molecules such as vitamin K.
Thus far it has been thought that the human intestinal cells do not interact with the microflora found there, as they have a natural barrier separating them from the bacteria. Nevertheless, an interaction can be defined in various ways; it could be a direct physical contact between the bacteria and the cells or it could be the cell’s uptake of substances secreted by the bacteria, for example sodium butyrate. Even though, as mentioned, important molecules are provided to the human body by the microbiota, the latter is still a fairly undiscovered territory.
In this project, we look into the interaction between bacteria and intestinal cells. In order to do so, Caco-2 cells are transfected with plasmids containing promoters of interest and treated with sodium butyrate, a compound secreted by bacteria in the intestine. The gene expression is then measured through the activity of reporter genes.
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THE INTESTINE
Epithelial Cells Epithelial cells are cells that form a sheet called epithelium that is found lining different organs, glands and the outer surface of our body such as the intestine, lungs or the mouth. These cells are found to have different shapes and functions. Depending on the organ and its location, epithelial cells can be arranged either in single or multiple layers. (Mannheim, 2014)
The single layer of cells called simple epithelium can be found, for example, in the intestine, blood vessels and sweat glands. Each cell has an apical surface that is facing into the open space as well as a basal surface that faces the extracellular matrix to which it is attached. This tissue – the basement membrane – is made of glycoproteins and collagen and allows the exchange of materials between the epithelial cell and the blood capillaries that lie underneath (Figure 1). This is the case in the intestine where molecules that have been broken down from food pass through the epithelium and basement membrane to reach blood vessels and are then transferred to the rest of the body. On the contrary, multiple layers of cells – stratified epithelium – are found, for example, on our skin where the top layer of cells can be replaced immediately by the one underneath once it is worn down. (Hill, 2012) Figure 1 – Epithelial cells with their apical and basal surface. The basement membrane separates them from the connective tissue, but also allows the exchange of materials. (The Epithelium, 2016) Shapes Usually, epithelial cells are divided into three groups depending on their shape. Squamous cells are flat and low, making their shape ideal for surfaces that require a flow of fluid such as blood vessels or surfaces, like in the lungs, that require a thin layer for molecules to pass
Page 6 of 64 through. Cuboidal epithelial cells form cubes in surfaces that usually absorb or secrete substances. Columnar cells are long and thin, usually found where mucus is secreted like the respiratory tract, stomach and intestine. Furthermore, some epithelial cells have microvilli which are thin and fingerlike extensions of the apical surface. These structures allow an increase in the area of contact of the epithelial cells for example with the content of the intestine. Finally, certain specialized epithelial cells can have hair-like projections called cilia that can move from side to side. These cilia can either facilitate the movement of fluid over the cells, like mucus in the respiratory tract or the intestine, or give the cell motility. (Stevens, 1997)
Junctions between the cells The structural feature of an epithelium is given by the connection that binds neighboring cells. These cell junctions can be separated into three groups according to their function: 1) Occluding junctions link the cells in order to create a barrier 2) Anchoring junctions provide strength and stability 3) Communicating junctions make the selective exchange of cell-signaling molecules between cells possible
In occluding junctions, also called tight junctions, cell membranes of neighboring cells fuse or meet usually just under the apical surface. This type of junction prevents molecules from diffusing between the individual epithelial cells as well as from one side of the epithelium to the other. Anchoring junctions occur at localized regions where glycoproteins from adjacent cells interlink in the space between them. Such a junction is called a desmosome and it ensures that the epithelial cells form a cohesive unit. Lastly, communicating junctions or gap junctions are also only found in certain places. At gap junctions, there are open pores in each of the adjacent cell membranes which are aligned and connected to each other. These pores are formed by six protein subunits – forming a connexon – and allow small molecules to pass through as there are no cell-membrane boundaries, allowing continuity between the cytoplasm of the cells (Figure 2). These gap junctions are important in cell-cell communication as they allow passage for signaling agents. (Hill, 2012)
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Figure 2 – The different types of junctions connecting epithelial cells. From left to right: gap junctions, desmosome and tight junctions. (Cell junctions, 2016)
Secretion of macromolecules Certain epithelial cells may have structural specialization that allow them to produce and secrete various macromolecules such as mucins, enzymes or steroids. Additionally, some of them can also produce and transport ions. All these secreted molecules are important for the proper functioning of the body. For example, mucins, which are made up of glycoproteins and proteoglycans, are found in different body cavities such as the mouth where they serve as a lubricant or in the stomach where they form a barrier. Some of the secretory cells can aggregate and form specialized glands that then have a focused production of the wanted secretion product. In many cases, these glands are distributed occasionally between non- secreting epithelial cells. (Stevens, 1997)
Epithelial cells have four mechanisms involved in secretion that are divided into two categories according to their mechanism of action: endocrine secretion and exocrine secretion. The first type of secretion releases the secreted product by endocytosis into the bloodstream through the basal surface and basement membrane. However, exocrine secretion is further separated into three groups. Merocrine secretion releases the product from the apical surface of the cell by exocytosis. Apocrine secretion keeps the product in the apical cytoplasm and a part of the cell is then pinched off with the product. Lastly, holocrine secretion requires the cell’s death in order for it to become the product. (Stevens, 1997)
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Bacteria in the Intestine It has been found that there is a great symbiotic relationship between various bacterial species and the human body. While the stomach is nearly sterile, intestines contain over a 1000 different bacterial species (Figure 3). It is said that bacterial cells outnumber human cells approximately by a factor of ten. Both beneficial and harmful bacteria co-exist in the intestine, but in a healthy individual, the beneficial bacteria will be dominant. (Mitsouka, 1996) The mutualistic beneficial bacteria have various important functions that they carry out in the intestine. Their role varies from digesting food, to synthesizing carbohydrates, vitamins and nutrients, as well as stimulating immune responses and protecting cells from invasions of pathogenic bacteria. (Reece, 2011)
Figure 3 – Intestinal lumen with the mucus layer containing the bacterial microflora. (Canny, 2008)
As mentioned above, the gut flora is important for many reasons. Bacteria have a function in the development of the mucosal immune system and take care of breaking down dietary carcinogens. They also contribute with many metabolic molecules such as vitamin K, B and short-chain fatty acids. The latter are the result of fermentation of nondigestible carbohydrates, a key source of energy in the colon, by colonic bacteria and they have a trophic effect on intestinal epithelial cells. An example of a short-chain fatty acid is butyrate, which is the major energy source for enterocytes. On the other hand, the absence of bacteria
Page 9 of 64 in the intestine can have several negative outcomes including the reduction of digestive enzymes activity, reduction of cell turnover and a defective cell-mediated immunity.
Recent studies have confirmed what has been known for a while – the most abundant bacterial type found in the intestine is of genus Bacteroides, which are anaerobic gram- positive cocci. The microflora is said to serve as a “central line of resistance” from potential invasions by pathogens (Figure 3). Adherent bacteria, that are not pathogenic, can prevent the attachment as well as entry of harmful bacteria in the epithelial cells. Commensal bacteria compete for nutrients in the intestine thus maintaining the environment and preventing overproduction of nutrients which could lead to the introduction of competitors that could potentially become pathogenic. Furthermore, some molecules that signal the presence of invaders for the immune system, such as lipopolysaccharides (LPS) or lipoteichoic acid, can be derived from the resident microflora of the intestine. In a case where a pathogen would deactivate the cell’s function to start transcribing certain genes that would normally be activated in the presence of a harmful intruder, the intestinal bacteria also have some mechanisms that enable them to activate these genes thus maintaining a normal cellular response.
Even though epithelial cells and the resident flora of the intestine serve as barriers to eventual pathogens, these have found ways to overcome the hostile environment and weaken the barrier function. Some can target structures such as glycoproteins and glycolipids that can serve as an attachment point for bacteria. Others can find a way to enter the epithelial cells from the apical surface in order to get to the basal part, for example by disrupting cell junctions or by cytotoxic injury. An example of a pathogenic bacterium is the enteropathogenic Escherichia coli that is the cause of severe diarrhea which remains responsible for a high number of children mortality. (Geraldine, 2008)
Sodium Butyrate The energy requirement of the colonic epithelium is primarily met by sodium butyrate, the conjugate base of butyric acid. Butyrate is produced when the intestinal microbiota ferments certain plant-derived carbohydrates (dietary fibers) that go through the small intestine undigested (Lupton, 2004) ‘’ […] such as cellulose and pectin.’’ (Guyenet, 2009) The energy that butyrate supplies the epithelial cells with is so vital, that in its absence, the cells will eventually die from autophagy (ingesting themselves). (Donohoe, 2011) Page 10 of 64
Further, research has showed that butyrate regulates the expression of intestinal specific genes linked to apoptosis, cell proliferation and differentiation. This regulation is important for maintaining the optimum environment for the colonic epithelium. (Daly, 2006) A specific study regarding more than 19 000 different genes showed that when treated with butyrate, the expression of 221 of those genes was found to be regulated. (Daly, 2006) In this regard, butyrate could be playing an important role in colorectal cancer, since unregulated apoptosis and cell proliferation are factors often related to cancer development.
Butyrate does not only supply the intestine with the energy that is required for the many important processes to take place and for regulating the intestinal gene expression, but it also provides numerous other functions, some understood in more detail than others. Firstly, butyrate is shown to have anti-inflammatory properties. Recent research uncovers that butyrate prevents or stops inflammation of the colon in two ways: 1. By inhibiting a small protein called Interferon gamma (IFNγ) involved in the intercellular signaling. The IFNγ starts a cascade that activates the Signal Transducer and Activator of Transcription 1 (STAT1), which then upregulates inducible Nitric Oxide Synthase (iNOS) that is known to induce inflammation as it regulates the vascularity of tissue. So when butyrate inhibits IFNγ, it prevents inflammation all together. (Zimmerman, 2012) 2. The same research shows that the activation of STAT 1 increases the infiltration of T- lymphocytes. This means that T-lymphocytes are infiltrating highly vascularized tissues, causing an inflammation in the area by putting pressure on the vessels. However, with the presence of butyrate, STAT 1 is inhibited. (Zimmerman, 2012) The research further shows that butyrate induces T-lymphocytes to undergo apoptosis. The combination of these two modes of action, inhibit the actual inflammation from taking place. (Zimmerman, 2012)
Furthermore, it has been found that butyrate can inhibit tumor growth in the intestinal tract by downregulating the Vascular Endothelial Growth Factor (VEGF). (Kumar, 2008) This growth factor is directly involved in inducing the production of new blood vessels in the intestine to reroute the blood when smaller vessels are blocked or a larger blood supply is needed to oxygenate the tissue. (Kumar, 2008) It is known that cancers cannot continue growing and spreading if they do not have a proper blood supply, which is why the neoangiogenesis (the development of new blood vessel in tumorous tissues) is important for Page 11 of 64 cancer progression. (Kumar, 2008) When butyrate downregulates VEGF, the cancer cells that are present in the colon are deprived of blood and cannot evolve.
’’At the molecular level, butyrate inhibits Sp1-DNA binding activity by promoting Sp1 protein dephosphorylation in EAT cells.’’ (Kumar, 2008) Ehrlich ascites carcinoma (EAT) cells are spontaneously arising in epithelial cancer cells that are adapted to abnormally large amounts of fluid in the abdominal cavity of rodents. (Queiroz, 2004) The Transcription Factor protein (SP1), which regulates gene expression, has also shown to contribute to the aggressiveness of numerous carcinomas (epithelial cancers). (Wie, 2004)
All the above mentioned strongly implies that butyrate is of great significance to the health, specifically in order to maintain the proper function of the colon and prevent it from acquiring any diseases that would compromise its function.
GENE REGULATION
Gene Transcription Our genes hold crucial information about our organism that is expressed in the form of specific proteins. In order to synthesize these proteins, various processes must take place. First of all, the gene has to be transcribed into a messenger RNA (mRNA) molecule. This happens when the double stranded DNA unwinds at the site of the gene, allowing the genetic sequence to be read by a RNA polymerase. One of the single strands of DNA acts as a template for the RNA polymerase that reads the sequence and creates a complementary nucleotide sequence that becomes the pre-mRNA. The whole process of transcription happens in 3 steps (Griffiths, 2015): 1. Initiation: The initiation of gene transcription is complex and requires the cooperation of many different proteins. Upstream from the specific gene that has to be transcribed, there is a region, called a promoter, which has specific sites recognized by various proteins. One of these sites is called the TATA box and is recognized by the TATA-binding protein (TBP). This protein is part of a type of general transcription factors (GTFs) that, once bound to the promoter, attract the RNA polymerase and help position it at the correct starting point. The TATA-binding protein, the GTFs and the RNA polymerase
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form the preinitiation complex (PIC) at the promoter site which the RNA polymerase dissociates from afterwards in order to start the elongation of the mRNA. 2. Elongation: After RNA polymerase recognizes the promoter and starts the initiation of the transcription, DNA continues to unwind as RNA polymerase reads the template strand and the mRNA grows from the 5’- end to 3’- end (Figure 4). 3. Termination: Transcription is ended when RNA polymerase has finished reading the sequence found at the termination site and the whole mRNA has been synthesized. This step is the least understood and depending on the type of RNA polymerase it can require different factors. The mRNA, once released, is further processed. (Griffiths, 2015)
Figure 4 – Elongation process during gene transcription. Double stranded DNA is unwound and the gene sequence of interest is read by RNA polymerase that transcribes it into mRNA. (RNA Polymerase, 2016)
Regulation of Gene Expression Although we have thousands of genes, they are not all constantly expressed. The expression of genes is regulated at many levels, mostly separated in two categories: the transcriptional and the post-transcriptional gene regulation. (Griffiths, 2015)
In the first case, transcription factors (TFs), which are regulatory proteins, bind to specific DNA regions (outside the protein-coding region) therefore directly or indirectly controlling the rate at which genes are transcribed. One group of regulatory proteins is called activators. As their name suggests, these make sure that the transcription of the gene is
Page 13 of 64 activated by helping RNA polymerase bind the initiation site. On the other hand, repressors bind the promoter consequently blocking RNA polymerase’s access and the gene then cannot be transcribed. Because of the large eukaryotic genome and the organization of the DNA into compact nucleosomes, forming chromatin, most of the genes are “turned off” most of the time. The binding of the transcriptional machinery is often not possible when the DNA is wound up around the histones, therefore structural changes are required to access the promoter by the intermediate of sequence-specific DNA-binding regulatory proteins. Certain transcription factors can bind to an enhancer sequence that can be located at a distance from the promoter. The binding of an activator to the enhancer sequence turns on transcription. (Griffiths, 2015)
Post-transcriptional gene regulation is mostly regulated by various short non-coding RNA molecules such as microRNA (miRNA) or small interfering RNA (siRNA) that are responsible for repression of gene expression which is called gene silencing. For example, miRNAs can interfere with the translation of mRNA into proteins, either by binding the mRNA and repressing the translation or by removing the poly(A) tail that protects it from degradation. (Griffiths, 2015)
Gene Regulation in the Intestine In order for genes to be expressed in the amount, time and place needed, they are regulated by various signaling pathways and transcription factors. Some of the regulatory mechanisms found to be especially important to the intestine are related to the Sox9 or CDX2 genes as well as to the Notch-1 pathway.
The Sox9 ((Sex Determining Region Y)-Box 9) is a transcription factor that represses CDX2 (Caudal Type Homeobox 2) induced differentiation. The CDX2 is a transcription factor especially important in the regulation of specific genes involved in the cell growth and differentiation of intestinal cells. (CDX2, 2016) The Cdx gene family includes Cdx1, Cdx2, and CDx4 that all control differentiation that affects the arrangement of intestinal epithelia. Research done on mice showed that when the mutated allele of the Cdx2 gene is heterozygous, it provokes the development of polyps (benign tumors) in the colon. (Richmond, 2010)
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Furthermore, a single point mutation in the protein product of the K-RAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene can cause colorectal cancer, which makes its regulating mechanism especially important, while ILK (Integrin Linked Kinase) has showed to be a co-activator of pancreatic cancer phenotypes. A study shows that ILK and K- RAS are associated, as a knockdown of one will have the opposite effect on the other. (Chu, 2015) This means that a knockdown of pancreatic cancer genes (by targeting the ILK gene) will induce the development of cancers associated with the K-RAS gene. In this way, the two genes are regulators of each other and are highly involved not only in the development of cancer but also in the its spreading. (Chu, 2015)
The Notch-1 signaling pathway is important in the development of the crypt, which is made up from important stem cell-like glandular and absorptive cells found in the intestine. (Keil, 2012) Since the Notch-1 protein is working extracellularly, its inhibitor - Numb - regulates the protein by having it transported into the cell, inducing the inactivation of Notch-1. (Giebel, 2012)
Another example of a regulating signaling pathway is Wnt/β-catenin signaling that controls differentiation of specific intestinal cells and the intestinal proliferation. It also maintains a perfect balance between cell proliferation and cell induced apoptosis. (Richmond & Breault, 2010) The NF-KB complex, acting as a transcription factor, is also another important factor acting on the regulation of the expression of various types of genes.
NF-ΚB protein complex Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) is a protein complex which acts as a transcription factor. The NF-KB complex is found in almost all cells, but is in its active state only in some such as B-cells (B lymphocytes), T-cells and different types of cancer cells. (Gilmore, 2011) When in the active state, these protein complexes control DNA transcription of genes encoding cytokines (small proteins used as signals). When these are expressed, they are involved in the activation of different important cell response such as immune responses, which is also indicated in its name since B-cells are a type of white blood cells. (Immunoglobulin(Ig), 2016)
The NF-KB induces cellular mechanisms that react on invasions with bacterial and viral antigens. A response to a detection of antigens in the blood stream would be to induce the B- Page 15 of 64 lymphocyte’s production of immunoglobulins (antibodies) for recognition of said antigens. In this process, an important component of the immunoglobulins are the kappa-light chains. (Igk, 2016) The NF-KB protein complex is enhancing the B lymphocyte’s production of the kappa-light-chain component.
When found in the cytosol, NF-KB is bound to its inhibitor IKBα and in most cells is only found in this form. However, NF-KB can reach an active state by one of two pathways - the canonical and non-canonical pathway - both having the same essential mechanism in the activation of NF-KB. This begins with the activation of IKB (IKK), a kinase that phosphorylates the inhibitor of IKBα and thus allows NF-KB to enter the nucleus. There, the NF-KB will bind to its coactivator and to RNA polymerase in order to transcribe its target genes into proteins. These finished proteins cause changes in the cell function in a specific way (Figure 5). (Gilmore, 2006)
Figure 5 - NF-KB signaling pathway. The figure shows how NF-KB, by the use of a signaling cascade, induces the transcription of its targeted genes. (NF-KB, 2016)
The NF-KB protein complex is divided into two groups: canonical and non-canonical NF-KB complexes. The latter cannot activate transcription. However, in some cases, they can form heterodimers with molecules of the first group, which then enables them to activate transcription. The canonical complexes are different from the second group since they have C-terminal domains that specialize in transcription activation. (Gilmore, 2011) Both groups
Page 16 of 64 have common key proteins making up the complex: p65(RelA) and p50. These are made up around a specific protein domain called the Rel Homology Domain (RHD). (Wolberger, 1998) Studies have shown that the phosphorylation of the RHD domain can regulate the expression of the specific genes that have NF-KB as a transcription factor. (Anrather, 2005) In this RHD domain, the N-terminal is a DNA recognition site, which means that this part interacts directly with the DNA bases, while the C-terminal is where the NF-KB binding site to IKBα is placed. (Müller, 1995)
As mentioned, the NF-KB is active in some kinds of cells, including cancer cells, due to the lack of downregulation of NF-KB. The regulation mechanism is to inactivate the IKB kinase, which would mean that the IKBα would not be phosphorylated and the NF-KB could not start transcription. In cancer types like colon, lymphoid, breast and prostate, the NF-KB is constantly activated and therefore constantly found inside the nucleus. (Gilmore, 2011) ‘’ In some cancers, this is due to chronic stimulation of the IKK pathway, […] Moreover, several human lymphoid cancer cells have mutations or amplifications of genes encoding Rel/NF-kB transcription factors (esp REL in human B-cell lymphoma) [...]’ (Gilmore, 2011)
Further research has shown that regulating or blocking NF-KB in cancer treatment can be a key to a reduction in cell proliferation. (Yamamoto, 2001) It is thought that the activation of Rel proteins is positively associated with multiple cancers and might protect the cancer cells from apoptosis. A regulation of NF-KB would also help in the inducement of apoptosis and essentially be an important component in cancer treatment. (Gilmore, 2011)
Wnt signaling pathway The Wingless-related integration site (Wnt) is a family of different signal transducing glycoproteins acting as ligands. The Wnt protein ligands bind to a G-coupled Frizzled receptor, that then induces a series of intracellular steps - a signaling cascade - and the response that occurs will affect cell proliferation, apoptosis, differentiation, etc. (Johnson, 2006) There are three different Wnt pathways, but only the canonical pathway is related to the regulation of gene transcription .
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The essential mechanisms of the canonical Wnt pathway The Wnt pathway involves two essential proteins: adenomatous polyposis coli protein (APC) and β-catenin. The APC protein is a tumor suppressor and controls the amount of chromosomes present in the nucleus after cell division while the β-catenin protein is associated with mechanisms such as cell proliferation and cell differentiation. (APC, 2016)
The Wnt pathway can either be in an active or inactive state, depending on whether the Wnt protein is present in the environment surrounding the cell or not. When the pathway is inactive, it means that the transcription of the Wnt targeted genes is turned off, whilst when active, transcription is turned on.
In the active state, the Frizzled receptor will bind the Wnt ligand while it simultaneously binds Axin and receptor related proteins/co-receptors (LRP) intracellularly. This will activate the disheveled protein (Dsh) that detaches the protein β-catenin from the APC. (APC, 2016) Once β-catenin is detached from APC, it can accumulate in the cytosol, enter the nucleus and induce gene transcription by attaching to the LEF-1/TCF transcription factors (Figure 6). (Preston, 2006) Transcription is stopped when the expression of Wnt targeted genes has met the cell’s requirements. This happens when the APC degrades β-catenin and therefore prevents it from inducing transcription.
Figure 6 - Wnt signaling pathway. A) shows the deactivated Wnt pathway while B) shows the presense of the Wnt protein and therefore the activated Wnt signaling pathway. (Preston, 2006)
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The Wnt pathway and its relation to cancer Since the Wnt signaling acts as an activator of the TCF/LEF-1 transcription factors, it also means that the levels of Wnt signaling will have a regulatory effect on them, essentially determining to what extent the Wnt targeted genes are expressed. (Eastman, 1999) This means that different levels of Wnt can have various consequences.
The Wnt protein has been related to cancer for a long time, as it was originally classified as a proto-oncogene. (Johnson, 2006) More specifically, mutations in either the APC protein or in β-catenin are found in colon cancer, both hereditary and non-hereditary cases. Furthermore, mutations in the APC protein are the cause of 80% of all colon cancer cases. (Johnson, 2006) Lastly, research has shown that: ‘’Some colorectal cancers associated with defective mismatch DNA repair have been shown to result from Axin2 mutations’’ (Johnson, 2006) This shows that Wnt signaling pathway can acquire mutations in several important proteins, increasing the risk of developing cancer.
THEORY BEHIND THE METHODS
Caco-2 cells - a model of colon cancer cells Colorectal cancer or colon cancer is one of the most diagnosed types of cancer, especially in the western world. (Brown, 2007) Even though genetic factors are of great significance when looking into the causes of colon cancer, a considerable number of cases arise sporadically due to environmental effects, rather than genetic tendencies. Furthermore, it is concluded that many colon cancer incidences result from carcinogenic compounds in food, since the compounds in the stool come in direct contact with the intestinal epithelium, where the colon cancer predominantly has its offset. (Adrouny, 2002) (Brown, 2007) The colon specific type of cancer is called adenocarcinoma - it is a result of the accumulation of genetic mutations that cause a loss of control over different processes in the cell like its ability to induce apoptosis. (Adrouny, 2002)
The chromosomal instability pathway is one of the most well defined causes of colorectal cancer and is related to mutations in the APC gene that is a tumor suppressor gene located on the 5th chromosome. The mutant APC ensures genetic stability, destroys tumor suppression activity and instead activates the c-MYC oncogene that contributes to unhinged cell
Page 19 of 64 proliferation and carcinogenesis. In addition, mutations occurring in the following genes may contribute to development of colorectal cancer: the K-RAS gene, the DCC gene (deleted in colorectal cancer) and the p53 tumor suppressors (Adrouny, 2002).
Caco-2 cells are human epithelial colorectal adenocarcinoma cells (of glandular origin that may form a mass), which have been widely used in various experimental procedures for about 20 years. (Sambuy, 2005) They are enterocytes (absorptive epithelial cells) that take up nutrients and other molecules from the intestinal tract. (Sambuy, 2005) The parental cell line is of a heterogeneous origin, meaning that different cells within one cell culture can have different morphology and phenotypes. (Sambuy, 2005) This further means that specific conditions will have an effect on what genes are expressed and on which alleles. When the cells are handled in a specific way (in the individual laboratory), it results in cell lines that have a very specific differentiation. Hence, within one tumor, cells with different phenotypes may be expressed. (Altschuler, 2010)
The main reason that Caco-2 cells are very applicable in procedures similar to the one presented in this paper is because they form a confluent monolayer of cells, which is a good model for mimicking the actual intestinal epithelial barrier. (Sambuy, 2005) Caco-2 cells have been used widely to examine how well pharmaceutics are absorbed. (Darnell, 2010) More specifically, they have been used in the examination of how long a drug will be active in the body by looking at the efflux systems in the cells, for example by looking at P- glycoprotein (P-gp), a transporter used in the efflux system. (Darnell, 2010)
Since, as mentioned, the Caco-2 cells are spontaneously differentiated depending on their conditions, it is very difficult to compare results from experiments done on Caco-2 cells between different laboratories. Regardless of whether the same laboratory procedures were applied, the experiments will be incomparable, given that the phenotypic and morphologic traits found within the cell cultures will very much so depend on the specific conditions that they have been subjected to. (Altschuler, 2010)
Experimental Analysis of Gene Expression Sometimes it can be difficult to set up an experiment that would be representative of the expression of specific genes in the human body. A more convenient way to perform such a study is by the use of a method where only the promoter region of a gene is investigated. This Page 20 of 64 is done by splicing it into a reporter plasmid. The plasmids are then introduced into the host cells, such as Caco-2 cells, where they are expressed.
There are many different reporter plasmid assays available on the market such as luciferase, β-galactosidase, green fluorescent protein (GFP) or GUS assay. The reporter plasmid consists of a promoter of interest, cloned into the vector containing a reporter gene (for example, the pGL4 vector containing the luciferase reporter). The reporter gene codes for a protein whose activity can be measured by using various assays. For this project, bioluminescence activity of the luciferase enzyme will be used as a reporter of the transfection of Caco-2 cells. The first step of this procedure is to produce the recombinant plasmid which is later transfected into the host cells and its reporter gene activity is then measured.
Cloning Plasmid vectors used for cloning consist of various structural elements (Wong, 2006) : Replication of origin: specific sequence at which replication is initiated. Cloning site: artificially constructed recognition sequences for restriction enzymes that lead to more convenient insertion of the foreign DNA. Promoter region: enables transcription and translation of the inserted gene sequence of interest. Markers: usually genes encoding antibiotic resistance, that monitor transfected cells. Non-transfected cells will not survive in a medium containing the antibiotics.
Restriction enzymes recognize and cut the sugar phosphate backbone of the DNA molecule at specific recognition sites. These restriction enzymes are used to cut out the DNA segment of interest and to open a vector where segment can be introduced. A specific enzyme, DNA ligase, is then used to join the two sequences together forming a recombinant plasmid (Figure 7). The vector transports the gene into the host cell, usually a bacterium, where it multiplies and produces identical copies of itself. The plasmids are later purified and used for further experiments, such as transfection.
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Figure 7 – Recombinant DNA production mechanism. Restriction enzymes cut the DNA sequence of interest and open the vector, the gene sequence of interest is inserted and then DNA ligase joins them together. (Biotechteam, 2014)
Transfection using Polyethylenimine Transfection is a procedure that introduces recombinant plasmids into cells by using chemicals. Polyethylenimine (PEI) is an example of a chemical used in transfection experiments. It is available in two forms: one very long and branched (~ 800kDa) and another linear and of low molecular weight (~25 kDa). The linear PEI is a much better transfecting reagent as it is more stable in the cytoplasm and has a lower cytotoxicity. PEI contains one positive nitrogen per monomer subunit that binds the negatively charged phosphate backbone of nucleic acids and forms positively charged complexes (polyplexes) (Figure 8). The polyplexes interact with the cell membrane and enter the cells by endocytosis. It is necessary to have a slight excess of PEI that increases the ionic strength in the endosomes and makes them swell. This will help the DNA to be released from the endosomes and lysosomes into the cytoplasm. Later, during the mitosis, when the nuclear membrane breaks down, foreign DNA is able to enter the nucleus and express the gene product. (Reed, 2006)
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Figure 8 - DNA/PEI introduction in to the host cells. PEI binds DNA and they make positively charged complexes, that are taken up by the host cells by endocytosis. Later, DNA is released from endosomes and enters the nucleus during cell division. The foreign DNA can then be expressed by the host cell. (Yuhe, 2011)
The plasmids introduced into the cells by transfection contain specific promoters of interest followed by reporter genes, whose activity can be measured if the promoter is activated.
Luciferase and β-galactosidase reporter genes assay
This project was based on luciferase and β-galactosidase reporter gene assays. The two reporters were used to normalise the results from the measured promoter activity. Using the Dual-Light assay system, luciferase activity was measured first, followed by quantification of β-galactosidase later. (Martin, 1996)
The luciferase reporter technology is based on the interaction between the luciferase enzyme and the luminescent substrate, luciferin, that emits light. The most commonly used bioluminescent reporter is the firefly luciferase which is efficient due to its sensitivity. The luciferase catalyses an oxidation reaction of luciferin when ATP and oxygen are added. That results in producing the light that is measured by the luminometer (Figure 9). (Allard, 2008) Similar to luciferase, the LacZ reporter gene, that codes for β-galactosidase, is used as
Page 23 of 64 an internal control to monitor variations in the transfection procedure. The transfected cells containing vectors with promoters and reporter genes are lysed and treated with reaction substrate that is called Galacton plus. It improves the utility of β-galactosidase by increasing the sensitivity of the assay. The substrate reaction is initiated by the Accelerator-II that raises the pH and provides the enhancer that increases the light intensity. Both luciferase and β- galactosidase assays can be performed on one cell lysate, which is why the Dual-light method is one of the most efficient and suitable method.
Figure 9 - Luciferase and β-galactosidase assay principle. Picture on the right) shows that light is emitted when luminescent substrate luciferin is oxidised by luciferase when ATP and oxygen are added. Picture on the left) shows that β-galactosidase cleaves Galacton plus and when accelerator is added, light reaction can be measured. (Luciferase Reporters, 2016) (β- Gal, 2016)
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METHODS
Before the actual experiment, certain preparations had to be done. The first step was to prepare DNA mixes that would later be introduced in the cells. Secondly, an optimization experiment was done to determine the most convenient conditions for the transfection experiment.
1. Preparation of DNA mixes Different already prepared plasmids with promoters and genes of interest were mixed with CMV lacZ plasmids as well as pSK+ empty vectors. The individual promoters of interest were picked based on relevancy to the project. The lacZ was used to monitor the transfection efficiency since β-galactosidase was measured later on. pSK+ was used as an empty vector to stabilize the DNA content in the mix. The table below shows the different concentrations mixed for the three components: Plasmid containing gene CMV LacZ pSK+ Total amount of of interest (ng) (ng) (ng) DNA (ng)
20 10 20 50
2. Cell culture Caco-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Life technologies) containing 10% fetal bovine serum, 100 units/mL of penicillin and 100µg/mL of streptomycin. The confluent cells were washed with 1x phosphate-buffered saline (PBS) (Gibco, Life technologies) and trypsinated. The cells were then counted on a hemocytometer, in order to determine a precise count of cells per milliliter.
3. Transfection procedure For the transfection procedure, after having counted the number of cells/mL, the cells were diluted to the required concentration. For each experiment, a control had to be included. Before the cells were seeded in the wells, the prepared DNA mixes were incubated with PEI for 1 hour. This step allows the plasmids to bind to PEI and later be taken up by the cells. The plasmids mixed with PEI were then incubated with the cells overnight in 5% CO2 at 37ᵒC. The next day, the medium was replaced with new medium containing the desired butyrate
Page 25 of 64 concentration and incubated for 24 hours. Control cells were maintained simultaneously on medium without butyrate.
4. Optimization experiment This step was performed in order to determine the optimal number of cells and the PEI concentration needed per well in the actual experiment, in order to obtain a successful transfection. Two plasmids were selected for this experiment – TOPflash and VTI1a – as they are commonly used in laboratory experiments. Four different concentrations of PEI were selected as well as four different numbers of cells per well. The following table illustrates the set-up: Cell number / PEI 5 000 10 000 20 000 40 000 5 000 10 000 20 000 40 000 concentration (µM) 0.5 1 Cells with TOPflash Cells with VTI1a 2 3
5. Butyrate experiment In this set-up, 10 000 cells were seeded in each well. After incubating with PEI, all 68 plasmids of interest (already mixed with lacZ and pSK+) were added on the cells, each in their respective well, and incubated. The next day, the medium was replaced with medium containing 10 mM of butyrate (Sauer, 2007) and incubated for 24 hours, while still maintaining the control in clear medium. Everything was done in duplicates that were not independent.
6. Measurement of results In order to measure the results, the media was removed from both plates and the cells were washed with 50µL 1x PBS. Once PBS was removed, the cells were lysed using 50µL of lysis solution (Tropix Lysis Solution, Applied Biosystems) with 0.5mM dithiothreitol (DTT).
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Plates were shaken for 5 minutes and 10µL of each sample were then transferred to a suitable plate that would show a contrast (white plate) in order to detect signals in the Glomax machine (96 microplate luminometer, Promega).
The luminometer then injected 25µL of buffer A (Dual-Light Reagent Buffer A, applied Biosystems) and 100µL of buffer B (Dual-Light Reagent Buffer B, Applied Biosystems) in each well and the luciferase activity was measured.
The plate was then incubated at room temperature for 1 hour before adding 100µL of accelerator in each well and measuring the β-galactosidase activity.
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RESULTS
Specific experimental conditions Optimization Cell solutions of 3.6 mL were prepared First, the optimization step was done in order to with different concentrations find out the optimal cell number and PEI corresponding to the cell number needed concentration. Even though the dual-light assay (shown in the table below). was done with luciferase and galactosidase 200µL of cell solution was added in each measurements, it was chosen to only check the well and then, 10µL of DNA mixed with β-galactosidase activity to interpret the PEI was added on top. optimization results as the LacZ plasmid is used to Cells were lysed in 50µL of lysis buffer, measure the transfection efficiency. TOPFlash and 10µL were used for measurements VTI1a plasmids were chosen for the experiment because they were the ones mostly used in the laboratory in previous experiments. The results are presented below:
Table 1. Results of β-galactosidase activity measurement of TOPFlash transfection into Caco-2 cells for optimization. TOPFLASH EXPERIMENT PEI concentration Cell number mM 5000 10000 20000 40000 0,5 2956 8185 43897 1200422 1 196755 833573 2091816 2042514 2 41137 536709 1227623 1600931 3 405360 882938 1156520 31915
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TOPFlash β-galactosidase activity 2500000
2000000
1500000 5000 cells 1000000
10000 cells Luminescence 500000 20000 cells 40000 cells 0 0,5 1 2 3 PEI concentration (µM)
Figure 10 – Variations of the TOPFlash β-galactosidase activity, in the transfection optimization experiment, according to different PEI concentrations and cell number.
Table 2. Results of β-galactosidase activity measurement of VTI1a transfection into Caco-2 cells for optimization. VTI1a EXPERIMENT PEI concentration Cell number mM 5000 10000 20000 40000 0,5 4447 12487 107668 2481463 1 1099915 827934 200693 2057349 2 290325 1484083 1777528 987624 3 60939 1111107 957356 88015
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VTI1a β-galactosidase activity
3000000
2500000
2000000 5000 cells 1500000 10000 cells 20000 cells Luminescence 1000000 40000 cells 500000
0 0,5 1 2 3 PEI concentration (µM)
Figure 11 - Variations of the VTI1a β-galactosidase activity, in the transfection optimization experiment, according to different PEI concentrations and cell number.
From Figure 10, we can see that for 20 000 cells and a PEI concentration of 1 µM, the expression of β-galactosidase is the highest. However, we can also see that at the same PEI concentration and 40 000 cells, the expression is not increased, which means that the system is probably oversaturated. From Figure 11, we can see that 20 000 cells with PEI concentration of 1 µM does not give a high expression. We can also see that the VTI1a expression decreases with an increase of PEI concentration, which could mean that it is more sensitive to PEI than TOPFlash.
Based on these results, it seems that the most convenient transfection conditions, to use in further butyrate experiment, are reached at 10 000 cells and a PEI concentration of 1 µM for both plasmids. In theory, it would also be adequate to use a greater number of cells but it was decided to keep the cell number on the lower side as 96 well plates were used to seed the cells and it was important not to over-saturate the system.
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Butyrate experiment Specific experimental conditions 100µL of media with cells was Luciferase and β-galactosidase activity were measured added in each well and then 10µL of in order to evaluate butyrate’s effect on the gene DNA mixed with PEI was added on expression in Caco-2 cells. However, it was noted that top. the β-galactosidase numbers were fluctuating. In order to The second day, the media was normalise and observe the transfection efficiency, the β- changed; 100µL of media containing galactosidase numbers would have to be similar as we 10mM of butyrate was added in each have the same amount of cells and DNA content in well. every sample. It was observed that butyrate probably had The third day, cells were lysed in an effect on the β-galactosidase activity (Figure 12). 50µL of lysis buffer, 10µL of this lysate were used for measurements.
Butyrate's effect on β-galactosidase 8000000
7000000
6000000
5000000
4000000
3000000 Luminescence 2000000
1000000
0 Non treated Treated with butyrate
Figure 12 – The results represent the average of the β-galactosidase activity of all the plasmids, non treated and treated respectively. It shows that butyrate had an upregulating effect on the expression of β-galactosidase.
From Figure 12, we can see that the β-galactosidase numbers cannot be used as the difference between the treated and non treated sets is huge, it seems that butyrate upregulated the β- galactosidase expression 7-fold. Therefore, the evaluation of butyrate’s effect on different promoters had to be done based on luciferase activity only. The plasmids containing different promoters were grouped into 4 groups - intestine, cancer, cellular mechanisms related or others – in order to organize the results more clearly.
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Intestine related genes 600000 Non treated 500000 Non treated
Treated with butyrate 400000
Treated with butyrate 300000
Luminescence 200000
100000
0
Figure 13 – Results from the luciferase measurement of all intestine related plasmids. The results are shown for the two set ups - both the non treated sets as well as butyrate treated sets.
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Cancer related genes 500000 Non treated 450000
400000 Non treated
350000
Treated with butyrate 300000 Treated with butyrate 250000
200000 Luminescence 150000
100000
50000
0
Figure 14 – Results from the luciferase measurement for all cancer related plasmids. The results are shown for the two set ups - both the non treated sets as well as butyrate treated sets.
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General cellular mechanisms related genes 1000000
900000 Non treated
800000 Non treated 700000
Treated with butyrate 600000
500000 Treated with butyrate
400000 Luminescence 300000
200000
100000
0
Figure 15 – Results from the luciferase measurement for all plasmid genes involved in general cellular mechanisms. The results are shown for the two set ups - both the non treated sets as well as butyrate treated sets.
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Other genes 600000 Non treated
500000 Non treated
Treated with butyrate 400000
Treated with butyrate 300000
Luminescence 200000
100000
0
Figure 16 – Results from the luciferase measurement for all other plasmid genes that are expressed in the intestine. The results are shown for the two set ups - both the non treated sets as well as butyrate treated sets.
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From Figure 13 to 16, we can see that the luciferase expression was very low for certain plasmids. Furthermore, sometimes we can observe a great variation between the two duplicates, either treated or not treated with butyrate. It was therefore decided to pick out only the results of the plasmids that would be at least 3-fold up or downregulated or, if not, that would have a certain relevance to the project. Also, when the numbers from the duplicates were deviating, they were not considered. The chosen results for the up or downregulated plasmids are presented in the table and graph below. (see Appendix for the other genes’ functions)
Table 3. Results of all the plasmids chosen to have a significant up/downregulation or relevance from each of the four groups. The function for each plasmid is included as well as the presence on the gene of the NF-KB binding site. Name of Non treated Treated with butyrate NF-KB Function the gene Set 1 Set 2 Set 1 Set 2 Protein coding gene, is thought to play a role in the CGN 284463 250486 524357 600653 formation and the regulation of tight junctions between cells forming permeable barriers
Cdx2 2842 1511 26183 17264 Marker of adenocarcinomas of intestinal origin Cdx2 4160 3095 27639 12712 Marker of adenocarcinomas of intestinal origin +enhancer Iron outflow (efflux) in order to meet required Heph 161271 192503 12528 8960 + homeostasis inside the cells. The gene may be important for the development of the liver, kidneys and the intestines. Encodes a HNF4 29502 25184 17874 26156 + nuclear transcription factor that can bind to DNA, Intestine related genes related Intestine controlling the expression of several genes.
Transcription factor (also controlled by HNF4 61437 57829 19182 26874 + transcription). Believed to be important in the +enhancer developement of the intestine, liver and kidney.
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Improves the expression of luciferase in cancer cells pGL3-Pro 3020 2339 103640 122783 with telomerase positive activity. Encodes a protein that acts as a tumor suppressor and acts as an antagonist to the Wnt pathway. Also APC 1211 1259 10303 21969 + plays a role in cell migration and adhesion, activation of transcriptiona and cell apoptosis.
Encodes a protein that acts as a tumor suppressor APC and acts as an antagonist to the Wnt pathway. Also 4164 4504 16794 27760 + +Enhancer plays a role in cell migration and adhesion,
activation of transcriptiona and cell apoptosis.
s Encodes for a histone hairpin binding protein, which represes transcription by binding to he promoter of HBP1 81756 92685 414638 480185 + the target genes. Is also important for the regulation of cell cycle as well as in the Wnt pathway. Plays role in embryonic development of skeleton SOX9 6139 7967 157109 165718 + and reproductive system. Contains TCF4 binding sites and measure Wnt TOPFlash 8521 5930 1239 2143 Cancer related gene related Cancer signaling pathway activity.
Codes for important transcription factor involved in TCF4 7087 6645 107259 68289 + the last step of the canonical Wnt signaling pathway.
Transcriptional regulator. Important in tumor YAP1+ 121081 159009 48502 66461 supression by regulating cell proliferation and the Enhancer inducing appoptosis. Transcriptional regulator. Important in tumor YAP1 20205 18989 37158 54888 suppression by regulating cell proliferation and the inducing apoptosis Genes Cell growth regulator that controls cell cycle G1 CDKN2D 14563 15482 64851 79819 related to progression.
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general Regulates signaling pathways that control cellular diverse cellular functions including cell mechanisms CDC42 1029 1326 6987 4302 + morphology, migration, endocytosis and cell cycle progression.Involved in epithelial cell polarization processes. Encodes tyrosine membrane protein playing active Oca2 brown 15325 18807 182436 237990 role in melanin synthesis and determine the blue or (maxi 1) brown eye and the skin color in mammalians. Encodes tyrosine membrane protein playing active Oca2 enh.- 17751 8003 154538 205459 role in melanin synthesis and determine brown eye brown
and the skin color in mammalians.
Oca2 enh.- Involved in skin pigmentation and acts as a 16995 17197 239694 253513 blue determinant of blue or brown eye color. Encodes serine peptidase inhibitor for HGF SPINT1+
Other genes Other 13531 11205 64663 73620 (Hepatocyte Growth Factor) activator in wounded Enhancer tissues Transcription factor regulating the developement of HOXB4 19170 14290 54723 65037 the organism (the placement of cells,etc.) Encodes miRNAs playing an important regulatory miR194a+ 71071 75538 42 20905 role in the expression of genes, they regulate Enhancer translation of mRNA into protein.
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Figure 17 – Luciferase expression of the chosen plasmids divided into their respective groups. Also shows the specific factor of upregulation (numbers in black) or downregulation (numbers in grey).
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From Figure 17, we can see that several genes were affected variously by butyrate. From the Table 3, we can also see that the functions of some of the genes can be related. From these results, we further try to speculate about butyrate’s effect on general gene expression and its effect on important cellular pathways.
DISCUSSION
In our results, we showed the β-galactosidase numbers we obtained from our experiment and it clearly showed that butyrate had a certain effect on the CMV promoter of the lacZ plasmid, as the set treated was upregulated 6-fold. Since the CMV promoter is a viral promoter, it could be compared to the SV40 viral promoter that was present in the pGL3 promoter plasmid. This plasmid was highly upregulated (43-fold). A possible explanation for these results could be that butyrate targets a transcription factor that is common for both viral promoter, thereby having the same effect on them - upregulation. A logical possibility would be the NF-KB transcription factor, as it is known that it helps viruses escape the T-cells of the immune system. (Santoro, 2003)
We further investigated how butyrate could act on the activation of NF-KB and found an article that provided a possible explanation: “Butyrate […] enhanced NF-κB activation induced by TNF-α or IL-1β in our reporter cells. As such, butyrate-producing bacteria stimulated NF-κB activity and a strong correlation has been found between bacteria CM butyrate concentration and NF-κB activity.” (Lakhdari, 2011) From this information, we hypothesize that NF-KB could be a direct target of butyrate. Therefore, we tried to find a relation between NF-KB and our other genes, which explains why we searched whether each one had its transcriptional binding site. We then noticed that mostly all the plasmids with this binding site seemed to be upregulated, just like the CMV and SV40 promoters. This could support the fact that butyrate would have an upregulating effect on the NF-KB related genes.
Previous research also shows the cross-regulation between NF-KB and Wnt signaling pathways. (Du, 2010) It was seen that most of the genes involved in Wnt pathway also have NF-KB binding sites. The genome library (see Appendix) was used to see whether the genes that had up/down regulation when treated with butyrate also have transcription factor binding sites for RELA which is the antibody used to detect NF-KB.
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Cancer Related Genes From the different groups that we divided our plasmids into, certain genes were found to be cancer related. We noticed that most of these genes have in common their relation to the Wnt pathway. Proteins that are involved in this pathway can acquire mutations that will greatly increase the risk of developing cancer. The Wnt activity, in our case, could be measured by the TOPFlash activity assay. This plasmid contains 8 binding sites for TCF4, which is a key transcription factor that induces the canonical Wnt signaling pathway. This essentially means that we are measuring the TCF4 activity when measuring TOPFlash. The family of TCF proteins is directly involved in the last step of the Wnt pathway, where β-catenin binds the TCF and this β-catenin/TCF protein complex then induces the transcription initiation of the Wnt targeted genes. TCF4 is upregulated significantly (13-fold), which could indicate that butyrate activates the Wnt pathway. However, TOPFlash is downregulated 4-fold which is contradictory to our expectations, as it would be suspected that an upregulation of TCF4 would equal an upregulation of TOPFlash. Furthermore, TCF4 also shows an association to the NF-KB pathway and could therefore indicate that the TCF4 transcription was amplified by a butyrate induced NF-KB upregulation. This also explains why TOPFlash shows an affinity for NF-KB binding.
Another gene, whose protein is involved in the Wnt pathway, is APC that also showed a 13- fold upregulation. APC, in a complex with other proteins, is known to bind β-catenin, which results in its degradation. However, the APC with its enhancer was only upregulated 5-fold. The expression of the genes with the enhancer was, of course, expected to be higher than without it. The difference is a possible indication that butyrate somehow affects the enhancer’s activity.
On the other hand, Wnt signaling also has to be negatively regulated because if it was active all the time, it could result in abnormal cell proliferation and cancer later. HBP1 and SOX9 are both genes that encode proteins involved in this “repressing mechanism”. SOX9, upregulated 23-fold, encodes a protein that phosphorylates β-catenin by binding it and the components involved in its destruction, therefore stopping the Wnt signaling pathway. HBP1, upregulated 5-fold, inhibits the Wnt pathway in multiple ways, one of which is the disruption of DNA and TCF4 binding, which means that Wnt targeted genes are not transcribed. Both
Page 41 of 64 genes code for proteins that are outside factors, meaning they are not a part of the pathway itself, repressing the Wnt signalling.
The last cancer related gene that showed a 3-fold upregulation was YAP-1. This gene codes for a protein that has the same regulating role in the HIPPO pathway as β-catenin in the Wnt pathway. (Hong, 2015) The HIPPO pathway is also a known cancer related pathway, as it regulated the size of organs and a deregulation of this pathway can result in tumor formation. (Harvey, 2013) We also observed a 3-fold downregulation for the YAP-1 and with its enhancer. In the results graph, we can clearly see in the non-treated set that the enhancer was working, but once treated with butyrate its activity seems to be stopped. We can see this on the two treated sets (with enhancer and without) because they have very similar numbers.
To sum up, we could say that butyrate regulates the Wnt pathway as it upregulates both the inducing factors of this pathway as well as the factors that suppress it.
Intestine Related Genes A very important gene related to intestinal cells is CDX2 that codes for a major transcriptional regulator of intestinal specific genes involved in cell growth and in the development of adenocarcinoma. This also explains why the other three intestinal related genes - CGN, Heph and HNF4 - all have a certain association to CDX2.
The results show a 10 times upregulation of CDX2 with the treatment of butyrate, while a 6 times upregulation is observed with its enhancer. Once again, butyrate seems to stop the enhancer’s activity. Since CDX2 is usually downregulated in colon cancer (Bai, 2003), the upregulation by butyrate seems to be a positive outcome for the prevention of cancer and general intestinal regulation. We can even see that the non-treated cells had a very low expression of CDX2, which would make sense since we have cancer cells. Butyrate then boosts the expression of this tumor suppressing gene. Moreover, HNF4 is directly related to CDX2 , since HNF4, together with GATA factors, act as partner factors. (Roman, 2015) The HNF4 promoter shows a downregulation, even though it is outside the range chosen (since it is only 1.5-fold downregulated) it was decided to include based on its functional importance. If we compare the results from the HNF4 downregulation with the ones of the HNF4 with enhancer downregulation, we can see that the latter is more downregulated (3-fold). This
Page 42 of 64 result seems to show that butyrate also inactivates the HNF4 enhancer’s activity. Furthermore, HNF4 is the same as CDX2 - they are both tumor suppressors - and a mutation in this gene will lead to a higher risk of developing cancer. In such a case, a downregulation for this gene could mean a lower risk of mutations.
Lastly, the CGN and Heph genes are both direct binding targets of the CDX2 transcription factor (Boyd, 2010). The CGN gene encodes a protein important in the formation and regulation of tight junctions between the intestinal epithelial cells, essentially forming the permeable barrier between the intestinal lumen and the blood stream. The results obtained showed a 2-fold upregulation, meaning that it does not belong in the set minimum range, but based on this gene’s significance to the intestine and its association with the CDX2, it was decided to acknowledge the result. The Heph gene encodes a protein especially important in the transport of iron from the intestinal lumen into the bloodstream. The transfection results showed a downregulation of 16-fold. These findings are controversial to what is expected from the Heph expression because, similarly to the CGN gene, Heph has shown to be a direct target of the CDX2 transcription factor. (Boyd, 2010) However, there is a relation between colon cancer and iron deficiency in the organism (San, 2015) and since butyrate is always produced in the intestine, it could explain why in our colon cancer (caco-2) cells there is a downregulation of Heph.
Other Genes The rest of our plasmids were either relevant for general cellular mechanism, CDX2 targets or highly expressed in the intestine.
CDC42, upregulated 5-fold by butyrate, codes for an effector protein that regulates signaling pathways that control various cellular functions and is also involved in epithelial cell polarization processes. We cannot really say what butyrate targets that then results in a higher expression. However, we found that this gene has a medium affinity for NF-KB binding, which could explain its upregulation. The protein product of CDC42 is involved in signaling pathways and cytokines (products of NF-KB induced transcription) are as well. In the complex signaling mechanisms, these two might somehow be related and therefore affected by butyrate similarly.
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CDKN2D, upregulated 5-fold by butyrate, is a protein coding gene that acts as a regulator that controls the cell’s G1 phase progression. It is difficult to say whether such an effect would have a positive or negative outcome on the cellular level as no other relevant research has been done that could be related to our project. In principle, if the gene product was downregulated, it may have a negative effect on the cellular growth. It could also have a negative outcome when upregulated if it induces a high level of cell proliferation, resulting in cell overgrowth. However, it is not possible to definitively say whether or not this is the case, a comparison to the expression and the effects would have to be done in order to understand this part of the mechanism.
The Oca2 gene is most common in inherited skin pigmentation disorders. This gene is expressed in most of the human body tissues, including the intestine. Mutations in the Oca2 gene cause albinism and melanoma. (Hawkes, 2013) We had three different Oca2 gene constructs included in our project. The results show that the Oca2 genes were highly affected by the addition of sodium butyrate to the growth medium. An upregulation of 12 to 14-fold was seen, but the mechanism behind such an effect is unknown. Also, it has not been published yet, but Oca2 is suspected to be a CDX2 target. (Jesper Troelsen)
MicroRNA 194a (miR194a) is a short, non coding RNA that is involved in post transcriptional regulation of gene expression. Preliminary studies have shown that miRNA could be overexpressed in some human cancers. Unfortunately, the role of this specific microRNA in cancer is not fully understood. (Valinezhad, 2010) There was a 7-fold downregulation of miR194a expression seen when treated with butyrate. If miR194a does play a role in cancer development and is downregulated by butyrate, the result obtained shows a possible positive effect of butyrate on post transcriptional regulation of gene expression in cancer.
SPINT1 is a protein coding gene that is expressed in the whole body, but strongly in the epithelium of the gastrointestinal tract. It is known to be a potent inhibitor specific for hepatocyte growth factor activator (HGFAC), that is thought to be involved in activation of growth factors in wounded tissues. Previous studies demonstrated that SPINT1 could potentially inhibit the action of trypsin-like serine proteinases, which are involved in carcinogenesis and metastasis. Downregulation of SPINT1 gene expression has been reported in part of the colon, renal cell and ovarian carcinomas. (Kataoka, 2009) There was a 6-fold Page 44 of 64 upregulation of the SPINT1 gene seen when treated with butyrate. As mentioned, the downregulation of SPINT1 is seen in several cancers so the upregulation caused by butyrate could have a positive effect in preventing the development of cancer.
HOXB4 is a protein coding gene that is said to the important as sequence-specific transcription factor that is involved in development of anterior-posterior axis. A 4-fold upregulation was seen when treated with sodium butyrate. HOXB4 is also a CDX2 target (not published yet). (Jesper Troelsen)
Limitations of the transfection procedure With every experiment come certain technical and biological limitations, as it was the case in our procedure also. In an experiment like ours, it is always hard to evaluate the efficiency of the transfection. We cannot see what is happening with the cells and how much DNA they are able to take up. When we mixed our DNA samples with PEI in order to introduce them in our Caco-2 cells, we could only assume that each cell should theoretically take up the same amount of foreign plasmids. The set-up was an artificial system and it doesn’t necessarily have to work the same way in the genome. The DNA introduced in the plasmid cannot be expected to act as an endogenous gene.
In our case, it was even harder to evaluate the efficiency of the transfection since we noticed a problem with our measurements of β-galactosidase. We of course didn’t expect that the butyrate we used to treat the cells would also affect the expression of what was to be our control and cause variation in the numbers. For this reason, we could only rely on our results from the measurements of luciferase.
Furthermore, when we prepared our DNA samples, we mixed our plasmids with lacZ vectors as well as with pSK+ empty vectors. We did so as it was the common procedure that our supervisor used for her experiments. However, we later realized that we did not need to add the empty vectors since we were adding the same amount of DNA mix in all the wells. Therefore, we might have had better or different results if we had omitted the empty vectors as they might have binded to the PEI where the other plasmids could have been. If only the the lacZ vectors and our plasmids of interest were present, maybe more of them would have been taken up by the cells and we might have observed higher numbers of expression.
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When we decided to use butyrate as our “treating agent”, we tried to search for the optimal concentration that we should use. It was however hard to find any information and so we used a concentration of 10mM based only on one article, where we found that such an amount should not be toxic for colon cells. If there was more literature and previous experiments that we could have based ourselves on, we may have obtained better results if we had chosen a different concentration. This was also the case for our optimization experiment where we chose to add 15mM of lithium chloride on our cells, based on one article we found.
Overall, we were doing a new set up and it was hard not to have any literature to compare to. It would have been reassuring to be able to read what other researchers may have done and what kind of results they obtained. However, we still managed to get high numbers and interesting results from our experiment, which we were not necessarily expecting.
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CONCLUSION
According to the results obtained from our transfection procedure, we were able to see that sodium butyrate had an effect on the expression of several genes. TCF4, APC, HBP1, SOX9, CGN, CDCX2, CDC42, CDKN2D, YAP-1, HNF4, SPINT-1, Oca2 and HOXB4 were upregulated while TOPFlash, Heph and miR194a were downregulated. The analysis of related transcription factors and pathways of these genes indicates that butyrate could have an effect on the regulation of the Wnt pathway and the NF-KB complex. Further, in the results it is noticeable that butyrate has an effect on enhancer activity, which could indicate that it has an inhibiting effect on the enhancement of genes. The experimental set-up applied in this project seems to be an efficient method for measuring the effect of butyrate on the gene expression. However, one of the reporter used in this project showed to be influenced by butyrate, which means that the results cannot be considered as conclusive.
On the basis of the butyrate induced gene expressions found, we can see that there is an interaction between the beneficial bacteria in the intestine and the intestinal epithelial cells. As seen in the multiple publications regarding the pathways possibly related in this matter, butyrate could potentially be a factor in the reduction of intestinal and cancer related issues. However, further experiments would have to be conducted in order to get more conclusive evidence on the mechanisms and pathways affected by butyrate, that are behind the upregulation of the specific genes.
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PERSPECTIVES
As the results obtained in this project were somewhat inconclusive, further experiments must be conducted in order to fully understand the mechanism behind the interaction between butyrate and the intestinal cells.
As the β-Gal was affected by butyrate, it is necessary to find a reporter plasmid that is not influenced by butyrate in order to normalize the results. To understand the effect of butyrate, an extend of incubation time would have to be applied. Using a NF-KB inhibitor would help in the conclusion of which genes are actually targeted by it and whether butyrate induces its activity. The experimental scale would have to be performed in independent triplicates, enabling us to confirm the results.
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REFERENCES
Books
Adrouny, R. (2002). Understanding Colon Cancer. Jackson, MS, USA: University Press of Mississippi. E-book: http://site.ebrary.com/lib/rubruc/reader.action?docID=10157864&ppg=31. Accessed: 03.04.2016.
Allard S. (2008). Luciferase reporter assays: powerful, adaptable tools for cell biology research. Cell notes. Page 23-26.
Brown, G. (2007). Colorectal Cancer. Cambridge, GB: Cambridge University Press. E-book: http://site.ebrary.com/lib/rubruc/reader.action?docID=10410324&ppg=15. Accessed: 03.04.2016.
Brown T. (2010). Gene Cloning and DNA Analysis: An Introduction (6). Hoboken, GB: Wiley-Blackwell.
Griffiths A, Et.al. (2015). Introduction to Genetic Analysis. 11th edition. New York, W. H. Freeman and Company.
Hill, Wyse and Anderson. (2012). ANIMAL PHYSIOLOGY – third edition. Massachusetts, USA: Sinauer editorial. Page 37-40.
Martin C. (1996). Dual Luminescence-Based Reporter Gene Assay for Luciferase and β- Galactosidase, Biotechniques. Page 520-524.
Reece, et.al. (2011) CAMPBELL BIOLOGY – ninth edition. San Francisco, USA: Pearson Education. Page 617.
Stevens A and Lowe J. (1997). HUMAN HISTOLOGY 2nd Edition. Harcourt Publishers Limited. Chapter 3.
Wong D. (2006). The ABCs of Gene Cloning - Second Edition.
Yuhe W. (2011). Branched Polyethylenimine Derivatives with Reductively Cleavable Periphery for Safe and Efficient In Vitro Gene Transfer. Biomacromolecules. Page 1032– 1040.
Articles
Altschuler S and Wu L. Cellular Heterogeneity: Do Differences Make a Difference? Cell journal, May 2010;141(4):559-563.
Anrather J, Racchumi G and Iadecola C. cis-Acting Element-specific Transcriptional Activity of Differentially Phosphorylated Nuclear Factor-KB. The Journal of Biological Chemistry, Jan. 2005;280(1):244-252.
Page 49 of 64
Bai Y, et.al. CDX2, a homeobox transcription factor, upregulated transcription of the p21/WAF1/CIP1 gene. The Oncogene Journal, Apr.2003;22(1):7942-7949.
Boyd M, Troelsen J, et.al. Genome-wide Analysis of CDX2 Binding in Intestinal Epithelial Cells (Caco-2). The Journal of Biological Chemistry, Aug. 2010; 285(1):25115-25125
Canny G and McCormick B. Bacteria in the Intestine, Helpful Residents or Enemies from Within? Journal of Infection and Immunity, Aug. 2008;76(8):3360-3373.
Chu, et.al. Regulation of oncogenic KRAS signaling via a novel KRAS-integrin-linked-kinase- hnRNPA1 regulatory loop in human pancreatic cancer cells. Journal of Oncogenesis, Nov.2015; E-published ahead of print.
Daly K and Shirazi-Beechey S. Microarray analysis of butyrate regulated genes in colonic epithelial cells. Journal of DNA Cell Biology, Jan. 2006; 25(1):49-62.
Darnell M, et. al. Investigation of the involvement of P-glycoprotein and multidrug resistance-associated protein 2 in the efflux of ximelagatran and its metabolites by using short hairpin RNA knockdown in Caco-2 cells. Drug Metabolisme Disposition, Mar. 2010; 38(3):491-497.
Donohoe R, et. al. The Microbiome and Butyrate Regulate Energy Metabolism and Autophagy in the Mammalian Colon. HHS Public Access - Cell Metabolism Journal, May 2011;13(5):517-526.
Du Q. and Geller D. Cross-Regulation Between Wnt and NF-KB Signaling Pathways. International Journal of Immunopathology and Pharmacology; Jan. 2010;1(3):155-181.
Eastman Q and Grosschedl R. Regulation of LEF-1/TCF transcription factors by Wnt and other signals. Current Opinion in Cell Biology, Apr. 1999;11(2):233-240.
Giebel B and Wodarz A. Notch Signaling: Numb Makes the Difference. Current Biology, Feb. 2012; 22(4):133-135.
Gilmore, T. Introduction to NF-kappaB: players, pathways, perspectives. Oncogene, Oct.2006;25(51):6680-6684.
Harvey K, et.al. The Hippo pathway and human cancer. Journal of Nature Reviews Cancer, Apr.2013;13(1):246-257.
Hawkes J, et.al. Report of a novel OCA2 gene mutation and an investigation of OCA2 variants on melanoma risk in a familial melanoma pedigree. Journal of Dermatological Science, Jan.2013;69(1):30-37.
Hiroshi I, et.al. Upregulation of HGF activator inhibitor type 1 but not type 2 along with regeneration of intestinal mucosa. American Journal of Physiology, Apr. 2000;278(4):635- 643.
Page 50 of 64
Hong A, Meng Z and Guan K. The Hippo pathway in intestinal regeneration and disease. The Journal of Nature Reviews Gastroenterology & Hepatology, May 2015; E-published ahead of print.
Johnson M and Rajamannan N. Diseases of Wnt signaling. Reviews in Endocrine and Metabolic Disorders, Jun.2006;7(1-2):41-49.
Keil, et.al. Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells. Development, Feb.2012; 139(3):488-497.
Kumar S, et.al. Butyrate-induced phosphatase regulates VEGF and angiogenesis via Sp1. Archives of Biochemistry and Biophysics, Oct. 2008; 478(1):85-95.
Lakhdari O, et.al. Identification of NF-KB Modulation Capabilities within Human Intestinal Commensal Bacteria. Journal of Biomedicine and Biotechnology, June 2011; E-published.
Lupton J. Microbial Degradation Products Influence Colon Cancer Risk: the Butyrate Controversy. The Journal of Nutrition, Feb. (2004);134(2):479-482.
Mitsouka T. Intestinal flora and human health. Asia Pacific Journal of Clinical Nutrition, Mar.1996;5(1):2-9.
Müller, et.al. Structure of the NF-KB p50 homodimer bound to DNA. Journal of Nature, Jan.1995;373(1):311-317.
Preston S, et.al. Mechanisms of disease: from stem cells to colorectal cancer. Nature Clinical Practice Gastroenterology & Hepatology, May 2006;3(5):267-274.
Queiroz M, et.al. Ehrlich ascites tumor as a tool in the development of compounds with immunomodulatory properties. The Journal of Immunopharmacology and Immunotoxicology, 2004;26(4):511-525.
Reed S. (2006). Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. Journal of Virological Methods, 138(1):85–98.
Roman A, et.al. Transcription Factors GATA4 and HNF4A Control Distinct Aspects of Intestinal Homeostasis in Conjunction with Transcription Factor CDX2. The Journal of Biological Chemistry, Dec.2015;290(1):1850-1860.
Sambuy Y, et.al. The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Journal of Cell Biology and Toxicology, Jan. 2005;21(1):1-26.
San R, et.al. Transcription factors GATA4 and HNF4A control distinct aspects of intestinal homeostasis in conjunction with transcription factor CDX2. Journal of Biological Chemistry, Jan.2015;290(3):1850-1860.
Santoro M, Rossi A and Amici C. NF-kappaB and virus infection: who controls whom. The EMBO Journal, June 2003; 22(11):2552-2560.
Page 51 of 64
Sauer J, Richter K and Pool-Zobel B. Physiological concentrations of butyrate modulate genes of oxidative and metabolic stress in primary human colon cells. The Journal of Nutritional Biochemistry, Nov.2007;18(11):736-745.
Valinezhad O, et.al. Mechanisms of miRNA-Mediated Gene Regulation from Common Downregulation to mRNA-Specific Upregulation. International Journal of Genomics, Aug.2010; E-published.
Wan-ling H, et.al. Availability and toxicity of Fe(II) and Fe(III) in Caco-2 cells. Journal of Zhejiang University Science B, Sep.2008;9(9):707-712.
Wie D, et.al. Celecoxib Inhibits Vascular Endothelial Growth Factor Expression in and Reduces Angiogenesis and Metastasis of Human Pancreatic Cancer via Suppression of Sp1 Transcription Factor Activity. Journal of Cancer Research, Mar. 2004; 64(6):2030-2038
Wolberger C. Combinatorial transcription factors. Elsevir, Oct.1998; 8(5):552-559.
Yamamoto Y and Gaynor R. Therapeutic potential of inhibition of the NF-KB pathway in the treatment of inflammation and cancer. Journal of Clinical Investigation, Jan. 2001;107(2):135-142.
Zimmerman M, et.al. Butyrate suppresses colonic inflammation through HDAC1-dependent Fas upregulation and Fas-mediated apoptosis of T cells. American Journal of Physiology - Gastrointestinal and Liver Physiology, June 2012;302(12):1405-1415.
Webpages
Gilmore, Thomas. (2011) NF-KB Transcription Factors. http://www.bu.edu/nf-kb/. Accessed: 16.05.2016.
Guyenet S. (2009). Butyric acid: an Ancient Controller of Metabolism, Inflammation and Stress Resistance? Whole Health Source – Nutrition and Health Science. http://wholehealthsource.blogspot.dk/2009/12/butyric-acid-ancient-controller-of.html. Accessed: 03.04.2016.
Kataoka H. (2009). SPINT1 (serine peptidase inhibitor, Kunitz type 1). Atlas of Genetics and Cytogenetics in Oncology and Haematology. http://atlasgeneticsoncology.org/Genes/SPINT1ID44384ch15q15.html. Accessed: 25.05.2016.
Mannheim J. (2014). Epithelium. MedlinePlus - Trusted Health Information for You. https://www.nlm.nih.gov/medlineplus/ency/article/002363.htm. Accessed: 25.05.2015.
APC - adenomatous polyposis coli. (May 2016) U.S. National Library of Medicine - Genetics Home Reference. https://ghr.nlm.nih.gov/gene/APC. Accessed: 19.05.2016.
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β-Gal Reporter Gene Assay, chemiluminescent. (2016). Life Science. https://lifescience.roche.com/webapp/wcs/stores/servlet/ProductDisplay?partNumber=3.5.3.1 8.6.2. Accessed: 26.05.2016.
CDX2 (2016). GeneCards Human Gene Database – The Weizmann Institute of Science. http://www.genecards.org/cgi-bin/carddisp.pl?gene=CDX2&keywords=cdx2. Accessed: 19.05.2016.
Cell Junctions. Wikipepdia. https://en.wikipedia.org/wiki/Cell%E2%80%93cell_interaction#/media/File:Cell_junctions.p ng. Accessed: 26.05.2016.
Igk - Immunoglobulin kappa chain complex [Mus musculus (house mouse)]. (May 2016) NCBI - Gene. http://www.ncbi.nlm.nih.gov/gene/243469. Accessed: 16.05.2016.
Immunoglobulin(Ig). (2016) affymetix - eBioscience. http://www.ebioscience.com/knowledge-center/antigen/immunoglobulin.htm. Accessed: 16.05.2016.
Luciferase Reporters. (2016). ThermoFisher Scientific. https://www.thermofisher.com/dk/en/home/life-science/protein-biology/protein-biology- learning-center/protein-biology-resource-library/pierce-protein-methods/luciferase- reporters.html#. Accessed: 26.05.2016.
NF-KB. (May 2016). Wikipedia - The Free Encyclopedia. https://en.wikipedia.org/wiki/NF- %CE%BAB. Accessed:16.05.2016.
RNA polymerase. Principle of Cell Biology. www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-21/21_09.jpg. Accessed: 26.05.2016.
The epithelium. SlideShare. http://www.slideshare.net/drshrikants/epithelium-in-general. Accessed: 26.05.2016.
SOX9 (2016). GeneCards Human Gene Database – The Weizmann Institute of Science. http://www.genecards.org/cgi-bin/carddisp.pl?gene=SOX9&keywords=sox,9. Accessed: 19.05.2016.
Plasmid table references
Richmond C and Breault D. Regulation of gene expression in the intestinal epithelium. Progress in Molecular Biology and Translational Science, 2010;96(1):207-229.
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Yee A and Paulson K. HBP1, Wnt Signaling and Breast Cancer.http://sackler.tufts.edu/Faculty-and-Research/Faculty-Research-Pages/Amy- Yee. Accessed: 17.05.2016.
Addgene: The non profit plasmid repository. http://www.addgene.org/. Accessed: 17.05.2016.
Genecards: Human Gene Database, Weizmann Institute of Science.http://www.genecards.org/. Accessed: 17.05.2016.
pGL3 Luciferase Reporter Vectors. (2016). Promega Corporation. https://dk.promega.com/products/reporter-assays-and-transfection/reporter-vectors-and-cell- lines/pgl3-luciferase-reporter-vectors/?activeTab=0. Accessed: 17.05.2016.
Uniprot. http://www.uniprot.org/uniprot/. Accessed: 17.05.2016.
TCF Reporter Plasmid Kit. Merck Millipore. http://www.merckmillipore.com/DK/en/product/TCF-Reporter-Plasmid-Kit,MM_NF-17- 285. Accessed: 17.05.2016.
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APPENDIX
Plasmids information:
Table 1. Intestine related genes and their functions. Plasmid Gene Names Function Number 166 SI 257 C sal (257 bp Sucrase Intestinal specific construct [provided by supervisor]. Isomaltase) + LCT enhancer (C version) 168 SI 257 T sal (257 bp Sucrase) + LCT Intestinal specific construct [provided by supervisor]. enhancer (T version) 336 CGN (Cingulin) Protein coding gene, is thought to play a role in the formation and the regulation of tight junctions between cells forming permeable barriers [3]. 342 TREH (Trehalase (Brush-Border The intestinal trehalse is thought to be envolved in hydrolizing trehalose (a dissacharid) Membrane Glycoprotein)) [3]. 382 ELMO3 (Engulfment And Cell Rearrangement of cytoskeleton, for the epithelia's motility, uptake of bacteria and Motility 3) inducement of phagocytosis [3]. 414 Heph (Hephaestin) Iron outflow (efflux) in order to meet required homeostasis inside the cells [3]. 418 PKP3 (Plakophilin 3) Abundant in the desmosomes of stratified epithelial cells, but absent in simple epithelial cells. It is also expressed in the colon and its tumors [2]. 464 CLDN2 (Claudin 2) Encodes protein expressed in the intestine, an integral membrane protein responsible for tight junctions [3]. 473 CLDN2 + enhancer Encodes protein expressed in the intestine, an integral membrane protein responsible for tight junctions [3]. 588 Long vCDX2 Alternative promoter of CDX2 [provided by supervisor]. 589 Short vCDX2 Alternative promoter of CDX2 [provided by supervisor]. 596 Long vCDX2 + intronic enhancer Alternative promoter of CDX2 [provided by supervisor].
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597 Short vCDX2 + intronic enhancer Alternative promoter of CDX2 [provided by supervisor]. 598 CDX2 (Caudal Type Homeobox 2) Marker of adenocarcinomas of intestinal origin [3]. 599 CDX2 + enhancer Marker of adenocarcinomas of intestinal origin[3]. 420 HNF4 (Hepatocyte Nuclear Factor 4) The gene may be important for the development of the liver, kidneys and the intestines. Encodes a nuclear transcription factor that can bind to DNA, controlling the expression of several genes [3]. 421 HNF4 (Hepatocyte Nuclear Factor 4)+ Transcription factor (also regulated by transcription). Believed to be important in the Cw-enhancer development of the intestine, liver and kidney [3].
Table 2. Cancer related genes and their functions. Plasmid Gene Names Function Number 180 Simian Virus 40-SV40 Improves the expression of luciferase in cancer cells with telomerase positive activity [1]. promoter 486 ST14 (Suppression Of Degrades extracellular matrix. Found in cancer cells and metastasis. Involved in the terminal Tumorigenicity 14) differentiation of keratinocytes [4]. 498 ST14 + enhancer Degrades extracellular matrix. Found in cancer cells and metastasis. Involved in the terminal differentiation of keratinocytes [4]. 509 APC (Adenomatous Polyposis Encodes a protein that acts as a tumor suppressor and acts as an antagonist to the Wnt pathway. Coli) Also plays a role in cell migration and adhesion, activation of transcription and cell apoptosis [3]. 510 APC + enhancer Encodes a protein that acts as a tumor suppressor and acts as an antagonist to the Wnt pathway. Also plays a role in cell migration and adhesion, activation of transcription and cell apoptosis [3]. 511 Axin2 Acts as an inhibitor of the Wnt pathway by down-regulating β-catenin (probably facilitates its phosphorylation) and is therefore probably a strong factor in different types of cancer[3]. 512 Axin2 + enhancer Acts as an inhibitor of the Wnt pathway by down-regulating β-catenin (probably facilitates its phosphorylation) and is therefore probably a strong factor in different types of cancer [3]. 514 HBP1(HMG-Box Transcription Encodes a histone hairpin binding protein, which represses transcription by binding to the Factor 1) promoter of the target genes. Is also important for the regulation of the cell cycle as well as in the Wnt pathway [3].
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545 LAMC2 (Laminin gamma 2) Subunit of laminin5. Involved in colon cancer metastasis [provided by supervisor]. 567 YAP1 + enhancer Transcriptional regulator. Important in tumor suppression: regulates cell proliferation and the inducement of apoptosis [3]. 568 YAP1 (Yes-Associated Protein Transcriptional regulator. Important in tumor suppression: regulates cell proliferation and the 1) inducement of apoptosis [3]. 644 SOX9 Transcription factor, repressor of differentiation, especially caused by CDX2 gene [7].
645 TOPFlash (LEF-1/TCF Contains TCF4 binding sites and measure Wnt signaling pathway activity. (Lymphoid enhancer binding factor 1)) 646 FOPFlash Upregulation of Thymidine Kinase (TK), with its mutated TCF (transfection grade T cell factor) binding sites provide negative control of TOPFlash. TopFlash encodes the wild type LEF1/TCF binding sites, while FOPFlash encodes a mutated LEF1/TCF binding site [6]. 650 GPA33 (Glycoprotein A33 Encodes a cell surface glycoprotein receptor (recognition antigen) expressed in more than 95% (Transmembrane)) of colon cancers. Involved in intercellular signaling [3]. 651 GPA33 + enhancer Encodes a cell surface glycoprotein receptor (recognition antigen) expressed in more than 95% of colon cancers. Involved in intercellular signaling [3]. 323 TCF4 (Transcription Factor 4) Codes for important transcription factor involved in the last step of the canonical Wnt signaling pathway.
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Table 3. Genes related to general cellular mechanisms and their functions. Plasmid Gene Names Function Number 258 MCM6(511) (Minichromosome Codes for protein that is essential for initiation of genome replication. Marker for Maintenance Complex Component 6 undifferentiated cells [3]. promoter (511 bp)) 259 MCM6(511) + LCT enhancer (C Codes for protein that is essential for initiation of genome replication. Marker for version) undifferentiated cells [3]. 260 MCM6(511) + LCT enhancer (T Codes for protein that is essential for initiation of genome replication. Marker for version) undifferentiated cells [3]. 321 H2B ( Histone) H2B is one of the subunits of a histone. Histones are responsible for DNA stability, repair, replication and transcriptional regulation. It seems that it may help the formation of a functional antimicrobial barrier in the epithelium of the colon [3].
325 MEP1A (Meprin A) Encodes proteins that are responsible for protein digestion and absorption, associated with glucose and insulin metabolism [3]. 326 LAMB2 (Laminin Beta 2 (LamininS)) Implicated in a wide variety of biological processes including cell adhesion, differentiation, migration, signaling, neurite outgrowth and metastasis. Involved in cancer pathways [3]. 368 CDKN2D (Cyclin-Dependent Kinase Cell growth regulator that controls cell cycle G1 progression [3]. Inhibitor 2D) 369 THAP (Domain Containing, Apoptosis DNA-binding transcriptional regulator, that regulates endothelial cell proliferation [1]. Associated Protein 1) 371 CDC42 (effector protein) Regulates signaling pathways that control diverse cellular functions including cell morphology, migration, endocytosis and cell cycle progression [3].Involved in epithelial cell polarization processes. 373 MAP3K2 (Mitogen-Activated Protein Codes for proteins of the serine/threonine kinase type. Which are involved in the Kinase Kinase Kinase 2) activation of other kinases involved in the MAP kinase signaling pathway and the FGFR (Fibroblast Growth Factor) signaling pathway [3]. 417 PURA (Purine-Rich Element Binding Encodes single-stranded DNA-binding protein which is sequence specific and effective
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Protein A) in DNA transcription and replication. If this gene is damaged, it could cause myelodysplastic syndrome and acute myelogenous leukemia [3]. 513 CK1 (Casein Kinase 1) Can phosphorylate many proteins. Participates in the Wnt pathway. Is also included in various cellular processes such as DNA repair, cell division and membrane transport[3]. 538 HOXA9 (Homeobox A9) Encodes homeobox genes and specific transcription factors.In the developmental stage helps to determine posterior and anterior axis of the cells [4].
Table 4. Other genes and their functions. Plasmid Gene Names Function Number 113 6 x HNF1A binding site Measure HNF1A binding CDX2 [provided by supervisor]. 221 LPH (Lactase Phlorizin hydrolase Controls LCT expression (lactase - hydrolyses lactose) [provided by supervisor]. 1044 bp) 222 LPH + LCT enhancer (T version) Controls LCT expression (lactase - hydrolyses lactose) [provided by supervisor].
223 LPH + LCT enhancer (C version) Controls LCT expression (lactase - hydrolyses lactose) [provided by supervisor].
354 MSI-1 (Musashi RNA-Binding Regulates translation of mRNAs. Is thought to have an influence on the proliferation and Protein 1) maintenance of neural stem cells [3]. 366 HNF1 (Homeobox A) Is a transcription factor required for the expression of several liver-specific genes. Can cause diabetes [3]. 378 Oca2 brown (Oculocutaneous Encodes tyrosine membrane protein playing an active role in the melanin synthesis. Albinism II) Determines blue or brown eye color and the skin color in mammalians [3]. 380 Oca2 + enhancer for brown eye color Encodes tyrosine membrane protein playing an active role in the melanin synthesis. Determines blue or brown eye color and the skin color in mammalians [3]. 381 Oca2 + enhancer for blue eye color Involved in skin pigmentation and acts as a determinant of blue or brown eye color [3]. 424 microRNA 194 + enhancer (in reverse Encodes miRNAs playing an important regulatory role in the expression of genes, they orientation) regulate translation of mRNA into protein [3]. 425 microRNA 194 + enhancer Encodes miRNAs playing an important regulatory role in the expression of genes, they
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regulate translation of mRNA into protein [3]. 500 SPINT1 (Serine Peptidase Inhibitor, Encodes serine peptidase an inhibitor of HGF (Hepatocyte Growth Factor) is an activator Kunitz Type 1) in damaged tissues [3]. 502 SPINT1 + enhancer Encodes serine peptidase an inhibitor of HGF (Hepatocyte Growth Factor) is an activator in damaged tissues [3]. 569 Basic reporter plasmid used in Involved in the mitochondrial electron transport chain. Electron carrier for FADH to pGL4.10 constructs coenzyme Q10 (Ubiquinone, Ubiquinol and Semiquinone) [3]. 579 SI200 (Sucrase-Isomaltase (Alpha- An enzyme vital for the last part of digestion of carbohydrates (sucrose) [3]. Glucosidase))(257 bp) 626 VTI1a (Vesicle Transport Through V-SNARE that mediates vesicle transport pathways through interactions with t-SNAREs Interaction With T-SNAREs 1A) on the target membrane. These interactions are proposed to mediate aspects of the specificity of vesicle trafficking and to promote fusion of the lipid bilayers [3]. 634 HOXB4 ( Homeobox B4) Transcription factor regulating the development of the organism (the placement of cells,etc.) [3].
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Raw data from results
Table 5. -galactosidase expression in intestine related genes.
Name of the Non treated Treated with butyrate promoter gene Set 1 Set 2 Set 1 Set 2 SI257-13910 C sal 9677 13897 3253 52460 SI257-13910 T sal 15462 16700 4403 61121 CGN 284463 250486 524357 600653 TREH 4991 1101 9193 13392 ELMO3 43939 49910 44572 115890 Heph 161271 192503 12528 8960 PKP3 374 41 47 70 CLDN2 50 52 89 57 CLDN2+Enhancer 40485 83727 13726 90 Long vCDX2 4511 454 1781 1910 Short vCDX2 57 47 144 132 Long vCDX2 330 65 215 139 +enhancer Short vCDX2 3767 93 427 450 +enhancer Cdx2 2842 1511 26183 17264 Cdx2 +enhancer 4160 3095 27639 12712 HNF4 29502 25184 17874 26156 HNF4 +enhancer 61437 57829 19182 26874
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Table 6. -galactosidase expression in cancer related genes.
Name of the Non treated Treated with butyrate promoter gene set 1 set 2 set 1 set 2 pGL3-Pro 3020 2339 103640 122783 ST14 7864 85 355 2313 ST14 +enhancer 10380 96 175 246 APC 1211 1259 10303 21969 APC+enhancer 4164 4504 16794 27760 Axin2 448 773 394 98 Axin2+enhancer 1409 1194 196 53 HBP1 81756 92685 414638 480185 Lam del3 2527 163 909 1314 YAP1+Enhancer 121081 159009 48502 66461 YAP1 20205 18989 37158 54888 SOX9 6139 7967 157109 165718 TOPFlash 8521 5930 1239 2143 FOPFlash 298 80 236 294 GPA33 63 70 297 342 GPA33+Enhancer 2262 1533 5881 5036 TCF4 7087 6645 107259 68289
Table 7. -galactosidase expression in cellular mechanisms related genes.
Name of the Non treated Treated with butyrate prmoter gene Set 1 Set 2 Set 1 Set 2 MCM6(511) 103363 105044 145505 2061 MCM6(511)-14C 99291 81045 186170 94691 MCM6(511)-14T 166343 166403 218549 213040 hH2B 195 43 106 26080 MEP1A 7027 7323 7001 298 LAMB2 267 114 478 55 CDKN2D 14563 15482 64851 79819 THAP1 1750 2208 1255 13248 CDC42 1029 1326 6987 4302 MAP3K2 1084 1336 2551 3026 PURA 200418 267099 618940 12405 CK1 191253 322903 488459 937694 HOXA9 2441 390 831 1108
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Table 8. -galactosidase expression in other genes.
Name of the Non treated Treated with butyrate promoter gene Set 1 Set 2 Set 1 Set 2 6xHNF (Cdx)- 33527 11096 6869 75196 Luc hLPH1044 6889 250 1209 39432 hLPH1044- 11332 8042 888 21191 13910T-sal endo hLPH1044- 4847 7306 911 6314 13910C-sal endo MSI-1 5032 1028 13366 29839 HNF1a 23445 35231 15667 12860 Oca2 brown 15325 18807 182436 237990 (maxi 1) Oca2 enh.-brown 17751 8003 154538 205459 Oca2 enh.-blue 16995 17197 239694 253513 miR194a+ 71071 75538 42 20905 Enhancer miR194a 11865 14896 3049 13161 +Enhancer SPINT1 6018 637 2986 8016 SPINT1+ 13531 11205 64663 73620 Enhancer pGL4.10 1820 614 1683 640 SI200 9839 890 37 93 VTI1a 387014 301757 533088 81796 HOXB4 19170 14290 54723 65037
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BIOINFORMATICS In order to check if specific genes that were affected by butyrate had NF-KB binding site (REL A), the genome bioinformatics data was collected. The example is shown below for SOX9 gene. The information was found on genome.uscs.edu. where we searched for the binding affinity for NF-KB for each respective promoter of interest.
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