HIF-1 Alpha-Induced Up-Regulation of miR-9 Contributes to Phenotypic Modulation in Pulmonary Artery Smooth Muscle Cells During Hypoxia

Pulmonary artery smooth muscle cells (PASMCs) are associated with the development of hypoxic pulmonary hypertension (HPH). Recent studies have implicated a critical role for microRNAs (miRNAs) in HPH; however, their expression and regulation in hypoxia-mediated phenotypic modulation of PASMCs remains largely unclear. Here, we report that miR-9 was induced in hypoxia and involved in a hypoxia-induced phenotypic switch in rat primary PASMCs. Knockdown of miR-9 followed by hypoxia exposure attenuated PASMCs proliferation and enhanced the expression of contractile genes in vascular smooth muscle cells (VSMCs), while overexpression of miR-9 in normoxia promoted a proliferative phenotype in PASMCs. The primary transcripts of miR-9-1 and miR-9-3, but not miR-9-2, increased dramatically after hypoxia, whereas silencing of the hypoxia-associated transcription factor HIF-1 alpha following hypoxia exposure abolished the enhancement of both primary transcripts in PASMCs. Using in silico analysis, we found three putative HIF-1 alpha binding motifs on miR-9-1 and one motif on miR-9-3 located within the 5-kb region upstream of the transcriptional start sites. Chromatin immunoprecipitation assay revealed that hypoxia enhanced the direct interaction between HIF-1 alpha and the regulatory elements of miR-9-1 and miR-9-3. Reporter assays showed that the regulatory regions of miR-9-1 and miR-9-3 behaved as enhancers in a HIF-1 alpha-dependent manner during hypoxia. Taken together, our data uncover a regulatory mechanism involving HIF-1 alpha-mediated up-regulation of miR-9, which plays a role in the hypoxia-induced phenotypic switch of PASMCs. (C) 2014 Wiley Periodicals, Inc.