Isoform-Specific Regulation of miRNA by HNF4alpha

摘要

Hepatocyte nuclear factor 4alpha (HNF4alpha/NR2A1) is a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors which plays a crucial role in the early development of the liver, intestine/colon, pancreas and kidney as well as maintaining metabolic and organismal homeostasis. Based on usage of alternative promoters (P1 and P2) and alternate splicing, it is estimated that nine different splice variants of HNF4alpha exist. The P1- and P2-driven variants differ by 16 amino acids in their N-terminal amino terminal (AB) domain that encodes an activation function (AF-1). It has been shown previously that expression of P1-driven HNF4alpha protein is decreased in different human cancers such as hepatocellular, gastric, renal and colorectal carcinomas, while P2-driven HNF4alpha is either unchanged or up regulated. HNF4alpha is known to play an important role during early embryonic colon development where it regulates genes related to epithelial function. In addition, HNF4alpha has been shown to function in protecting the epithelium during experimental colitis in young adult mice. The functional consequences of the up regulation of the P2-driven HNF4alpha in human cancer are not known.;In Chapter 2 a potential link between levels of specific splice variants of HNF4alpha and miRNA 21, an oncogenic miRNA is investigated. miR-21 has been implicated in the development, growth, progression and metastasis of different types of cancers including colorectal, hepatocellular and pancreatic cancers. A series of in vitro assays using human colorectal cancer cells are performed, miRNA-21 levels is detected using stem loop RT-PCR. The results show that ectopic expression of P2-driven HNF4alpha preferentially regulates miRNA 21 in human embryonic kidney and colon cancer cell lines that do not express endogenous HNF4alpha. Knockdown of P2-driven HNF4alpha in a colon cancer cell line that expresses endogenous HNF4alpha, results in preferential decrease of miRNA 21 levels in a different human colorectal cancer cells (Caco-2). In order to elucidate the potential functional significance of miRNA-21 regulation by HNF4alpha, both P1- and P2-driven HNF4alpha were knocked down using siRNA, This results in downregulation of miRNA 21 as well as reduction in cell number. Ectopic expression of a miRNA 21 mimic in the HNF4alpha knockdown cells partially restored the cell count. The molecular mechanism by which HNF4alpha regulates miRNA 21 expression is explored in experiments that show that HNF4alpha binds the promoter region of miRNA 21 and that P2-driven HNF4alpha preferentially activates the human miRNA 21 promoter in a transient transfection system. Taken together, these observations lead us to propose that P2-driven HNF4alpha preferentially activates miRNA 21, an oncogenic miRNA, thus suggesting a functional significance of P2-driven HNF4alpha upregulation in colon cancer.;In Chapter 3, the potential functional role of isoform-specific HNF4alpha and miRNA 21, in a mouse model of chemical (DSS) induced colitis is investigated in which colitis is induced by the addition of DSS in the drinking water. Mortality, rectal bleeding, colon length, and spleen/body weight ratios were all differentially affected in both male and female transgenic mice (expresses only P2- driven HNF4alpha) compared to WT indicating an enhanced susceptibility of the transgenic mice to DSS. Immunoblotting and stem loop RT PCR performed on colonic tissue isolated from the DSS-treated mice showed that the expression of both HNF4alpha and miRNA 21 correlates with better recovery from DSS-induced colitis. These findings suggest that HNF4alpha-mediated regulation of miRNA 21 may play an important role in colitis and could be used as a marker for improved prognosis of colitis.;In Chapter 4, a potential link between specific splice variants of HNF4alpha and miRNA 194-2 are explored. miR-194-2 has been implicated in intestinal epithelial differentiation, cell invasion and inhibition of Epithelial-Mesenchymal-Transition (EMT). Ectopic expression of P2-driven human HNF4alpha resulted in a preferential upregulation of miRNA 194-2 in human embryonic kidney and colon cancer cell lines. miRNA 194-2 levels were also found to be elevated in the distal colon of transgenic mice that expresses only P2-driven HNF4alpha. These results suggest that miRNA-194-2, like miRNA-21, may be preferentially regulated by P2-driven HNF4alpha. Further investigation is required to address the potential functional significance of this regulation.;Finally, in Chapter 5 the implications of the results of Chapters 2, 3, and 4 are discussed in detail. The importance of the role of HNF4alpha in general regulating these two microRNAs (miR-21 and miR 194-2) is considered as well as the relevance of the differential expression by the P1- and P2-driven isoforms. Finally, the role of these microRNAs and HNF4alpha splice variants are discussed in the broader context of the function of the colon in both the normal and diseased states.