MICRORNA-195 REGULATES PANETH CELL FUNCTION BY ALTERING SOX9 EXPRESSION VIA INTERACTION WITH RNA-BINDING PROTEIN HUR

Paneth cells reside at the bottom of the crypts in the small intestine and are crucial for maintaining homeostasis of the epithelium by engendering host protection from enteric pathogens. Defects in Paneth cells compromise the intestinal epithelial host defense and lead to a range of mucosal pathologies. MicroRNA-195 (miR-195) is evolutionally conserved among different species and it regulates the stability and translation of target mRNAs and is involved in many aspects of cell processes. MiR-195 can interact with RNA-binding proteins including HuR to jointly regulate target gene expression synergistically or antagonistically. We recently reported that intestinal epithelial tissue-specific transgenic expression of miR-195 resulted in Paneth cell dysfunction in mice, but the exact mechanism underlying miR-195 in the regulation of Paneth cell function remains unknown. Sox9 is a specific transcription factor essential for Paneth cell differentiation and its deletion stops Paneth cell development. Here we tested the hypothesis that miR-195 regulates Paneth cells by altering Sox9 expression via interaction with HuR. Methods: Studies were conducted in our newly generated miR-195 transgenic (miR195-Tg) mice, HuR knockout (IE-HuR-/-) mice, and cultured HEK cells. Paneth cells were examined by lysozyme-immunostaining assays. The functions of miR-195 and HuR were determined by their gene silencing or overexpression in vitro. Levels of Sox9 mRNA and protein were measured by qPCR and Western blotting analyses. The interactions of Sox9 mRNA with HuR or miR-195 were examined by RNP or biotin immunoprecipitation (IP)/qPCR analysis. Results: miR195-Tg mice exhibited reduced levels of Sox9 protein (by ~70%) in the small intestinal mucosa, which was associated with a significant decrease in the number of Paneth cells. Ectopically overexpressed miR-195 in HEK cells also decreased cellular abundance of Sox9 primarily by inhibiting Sox9 translation since miR-195 overexpression decreased the levels of newly synthesized Sox9 protein without effect on its mRNA content. In contrast, HuR overexpression increased Sox9 expression in HEK cells. IP/qPCR analysis revealed that miR-195 did not directly bind to the Sox9 mRNA but prevented HuR association with Sox9 transcript. Ectopically expressed HuR rescued Sox9 expression in cells overexpressing miR-195. Double mutant mice bearing a miR-195 transgene and HuR ablation were generated by crossbreeding miR195-Tg mice and IE-HuR-/- mice and displayed further decreases in the levels of Sox9 and Paneth cells in the small intestine. Conclusions: These results indicate that 1) miR-195 inhibits Sox9 translation through interaction with HuR; and 2) control of Sox9 expression by miR-195 and HuR plays an important role in Paneth cell development and function.