1114 Competitive Binding of Stim1 3′-Untranslated Region to RNA-Binding Protein HuR and MicroRNA-195 Regulates Its Expression and Epithelial Restitution

Abstract

Stromal interaction molecule-1 (STIM1) acts as a store Ca2+ sensor in intestinal epithelial cells (IECs) and plays an important role in the restitution after injury, but the exact mechanism that regulates STIM1 abundance remains unknown. mRNA turnover and translation are major posttranscriptional processes of gene expression and are governed primarily by RNAbinding proteins (RBPs) and microRNAs (miRNAs). HuR is a RBP that associates with labile mRNAs bearing AU-rich elements and regulates the stability and translation of its target transcripts. Several studies show that RBPs and miRNAs can function jointly in the regulation of a shared target mRNA. Based on computationally predicted HuR and miRNA-195 (miR195) associations with the STIM1 mRNA, this study tests the hypothesis that HuR and miR195 jointly regulate STIM1 expression in IECs and therefore the epithelial restitution after injury. Methods: Studies were conducted in differentiated IECs (IEC-Cdx2L1 line) induced by stable Cdx2-transfection. The interaction of HuR and STIM1 mRNA was detected by biotin pull-down and RNP-IP assays, whereas miR-195 binding to the STIM1 transcript was examined by RNA pull-down assay using biotin-labeled miR-195. The stability of STIM1 mRNA was examined by measuring its half-life with real-time PCR analysis. HuR and miR-195 functions were investigated by siRNA silencing and ectopic gene overexpression. Epithelial restitution was measured by examining cell migration after wounding. Results: STIM1 mRNA is a target of HuR and miR-195, and both HuR and miR-195 directly bound to the STIM1 3'-UTR but not to its the coding region or 5'-UTR. Mapping experiments and studies using heterologous reporter constructs revealed that there were significant overlaps between HuRand miR-195-binding sites on the STIM1 3'-UTR. HuR silencing reduced HuR/STIM1 mRNA complex, increased the levels of STIM1 transcript associated with miR195, destabilized STIM1 mRNA (by ~50%), thus inhibiting STIM1 expression. Ectopic HuR overexpression increased its binding to the STIM1 mRNA, decreased the abundance of miR195/STIM1mRNA complex, rendered the STIM1mRNA stable, and increased STIM1 protein. In contrast, overexpression of a miR-195 precursor increased miR-195/STIM1 complex, reduced HuR association with STIM1 mRNA, and destabilized STIM1 mRNA, thereby decreasing STIM1 expression levels. miR-195 overexpression also inhibited epithelial restitution in an In Vitro model as indicated by decrease in cell migration after wounding, which was prevented by HuR overexpression. Conclusions: These results indicate that 1) HuR and miR-195 are novel regulators of STIM1 mRNA stability and 2) competitive binding of HuR and miR-195 to the STIM1 3'-UTR regulates STIM1 expression and intestinal epithelial restitution after injury.