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. Page 2 of 64 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 Page 3 of 64 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. Page 4 of 64 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. Page 5 of 64 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.