Department of Pathophysiology and High Altitude Physiology, College of High Altitude Military Medicine, Chongqing, China; Key Laboratory of High Altitude Medicine, Ministry of Education, Chongqing, China; Key Laboratory of High Altitude Medicine, PLA, Third Military Medical University, Chongqing, China.
J Cell Physiol. 2014 Oct;229(10):1511-20. doi: 10.1002/jcp.24593.
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-1α following hypoxia exposure abolished the enhancement of both primary transcripts in PASMCs. Using in silico analysis, we found three putative HIF-1α 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-1α 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-1α-dependent manner during hypoxia. Taken together, our data uncover a regulatory mechanism involving HIF-1α-mediated up-regulation of miR-9, which plays a role in the hypoxia-induced phenotypic switch of PASMCs.
肺动脉平滑肌细胞 (PASMCs) 与低氧性肺动脉高压 (HPH) 的发展有关。最近的研究表明 microRNAs (miRNAs) 在 HPH 中起关键作用；然而，它们在低氧介导的 PASMCs 表型调节中的表达和调节仍很大程度上不清楚。在这里，我们报告 miR-9 在低氧诱导下被诱导，并参与大鼠原代 PASMCs 中的低氧诱导的表型转换。miR-9 敲低后暴露于低氧环境可减弱 PASMCs 的增殖，并增强血管平滑肌细胞 (VSMCs) 中收缩基因的表达，而在常氧条件下过表达 miR-9 可促进 PASMCs 的增殖表型。miR-9-1 和 miR-9-3 的初级转录物，但不是 miR-9-2，在低氧后显著增加，而低氧暴露后抑制缺氧相关转录因子 HIF-1α 则消除了 PASMCs 中这两种初级转录物的增强。通过计算机分析，我们在 miR-9-1 的转录起始位点上游的 5-kb 区域内发现了三个可能的 HIF-1α 结合基序，在 miR-9-3 上发现了一个基序。染色质免疫沉淀试验显示，低氧增强了 HIF-1α 与 miR-9-1 和 miR-9-3 的调节元件之间的直接相互作用。报告基因试验表明，miR-9-1 和 miR-9-3 的调节区在低氧条件下以 HIF-1α 依赖的方式作为增强子发挥作用。总之，我们的数据揭示了一种涉及 HIF-1α 介导的 miR-9 上调的调节机制，该机制在 PASMCs 的低氧诱导的表型转换中起作用。
