The small intestinal epithelium is constantly undergoing self-renewal. Intestinal epithelial cells migrate from the crypts, the site of cell proliferation, to the villi, where terminally differentiated absorptive enterocytes and secretory cells function. microRNAs (miRNAs) are a relatively new class of regulatory RNAs essential for cell proliferation and differentiation in all animal cell types and tissues studied. miRNAs generally repress gene expression by binding the 3'-untranslated region (3'UTR) of target mRNAs. I have identified 12 miRNAs that are differentially expressed in villi vs. crypts of the mouse small intestine. These miRNAs may play a role in regulating proliferation and differentiation of intestinal epithelial cells. For my first Aim, sythetic [sic] miRNA mimics and inhibitors will be used to test the ability of these miRNAs to alter intestinal cell growth and maturation in vitro. Also, I found that the crypt-enriched miR-152 is predicted to target Klf4, a transcription factor expressed in differentiated intestinal epithelial cells. Therefore, in my second Aim I will determine if miR-152 represses Klf4 expression with miRNA mimics and inhibitors in intestinal cell lines, and with a luciferase reporter construct that has the 3'UTR of Klf4 cloned downstream from luciferase. Additionally, the villus-enriched miR-22 is highly expressed in the small intestine. Through a collaboration, a miR-22(-/-) mouse is available to me to study miR-22 function in the intestine.
In Aim 3, I will look for mRNA targets of miR-22 in villus and crypt cells by expression array. These data will be combined with in silico predictions to help identify miR-22 targets. miR-22(-/-) mice will also be crossed with Apc(min/+) mice, a model of colorectal cancer, to determine if loss of miR-22 is tumorigenic. These data and techniques will form the basis for my independent research program to investigate mechanisms of miRNA-mediated gene repression in the small intestine. My mentors Dr. Douglas Burrin (intestinal physiology) and Dr. George Calin (miRNA biology), and the members of my scientific advisory committee, including Dr. Susan Henning, are experts in areas relevant to my proposal. The training I receive through this award will enable me to develop the skills and expertise needed for a career in biomedical research.
This work will advance our understanding of gene regulation in the intestine. Small RNA molecules are already being tested as therapeutic agents. Thus, knowing the miRNAs important for intestinal epithelial cell proliferation and differentiation and identifying their targets could help us find new treatments for intestinal diseases such as intestinal bowel disease and cancer, and for promoting intestinal regrowth and repair.
