Abstract 10829: Estrogen Regulation of miRs Expression in the Vasculature
Estrogen (E2) directly regulates the biology and function of cells and tissues within the cardiovascular system partially by activating E2 receptor alpha (ERα) and beta and regulating target genes expression. Many of the genes regulated by E2/ER in the vasculature are repressed yet the molecular mechanisms are still unclear. miRs are small non-coding RNAs that repress gene expression at the post-transcriptional level. We hypothesized a new gene regulatory network of E2 signaling in the vasculature in which E2 regulates miRs that in turn are responsible for repression of target genes. miR profiling analysis in aorta of overiectomized mice treated ex vivo with E2 for 4 h showed that E2 significantly regulated 32 miRs (26 up-regulated and 6 down-regulated). miR-203, one of the significantly up-regulated miRs, was further studied in cultured vascular smooth muscle cell (VSMC). Transient transfection assays showed that E2 induction of miR-203 was mediated by ERα and occurred at the transcriptional level. Deletion analysis of miR-203 putative promoter using reporter assay demonstrated that a documented ERα binding site was required for E2 regulation of miR-203. Site-directed mutagenesis using a reporter assay, of two binding sites in the miR-203 promoter for Zeb1, a transcription factor previously shown to repress miR-203 expression, and Zeb1 knockdown assay showed that Zeb1 contributes to E2 up-regulation of miR-203 by binding to the miR-203 promoter. Co-IP and ChIP assay revealed that ERα interacts with Zeb1 at the miR-203 promoter and E2 binding leads to disassociation of the ERα/Zeb1 complex from the miR-203 promoter. Target genes analysis showed that E2 repression of Abl-1 and p63 was mediated by miR-203. Over-expression of miR-203 in VSMC inhibited cell proliferation in vitro (21±3%, 25±1% and 26±5% inhibition on day 2,3 and 4, respectively). E2 inhibition of VSMC proliferation was diminished by miR-203 knockdown (17±7% p=0.019 v.s. 4±2% p=N.S. inhibition on day3). In conclusion, these findings provide the first evidence that E2 selectively regulates miRs that in turn contribute to repression of target gene in vascular cells and are indicative of an important role of miR-203 in E2-mediated inhibition of VSMC proliferation.
- © 2011 by American Heart Association, Inc.