Zhang Yanwei, Xu Jing
Department of Cardiac Surgery, the First Affiliated Hospital of Zhengzhou University, PR China.
Department of Cardiac Surgery, the First Affiliated Hospital of Zhengzhou University, PR China.
Biochem Biophys Res Commun. 2016 Apr 22;473(1):342-348. doi: 10.1016/j.bbrc.2016.03.116. Epub 2016 Mar 26.
miR-140-5p is down-regulated in patients with pulmonary arterial hypertension (PAH) and experimental models of PAH, and inhibits hypoxia-mediated pulmonary artery smooth muscle cell (PASMC) proliferation in vitro. Delivery of synthetic miR-140-5p prevents and treats established, experimental PAH. DNA methyltransferase 1 (Dnmt1) is up-regulated in PAH associated human PASMCs (HPASMCs), which promotes the development of PAH by hypermethylation of CpG islands within the promoter for superoxide dismutase 2 (SOD2) and down-regulating SOD2 expression. We searched for miR-140-5p targets using TargetScan, PicTar and MiRanda tools, and found that Dnmt1 is a potential target of miR-140-5p. Based on these findings, we speculated that miR-140-5p might target Dnmt1 and regulate SOD2 expression to regulate hypoxia-mediated HPASMC proliferation, apoptosis and differentiation. We detected the expression of miR-140-5p, Dnmt1 and SOD2 by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assays, respectively, and found down-regulation of miR-140-5p and SOD2 and up-regulation of Dnmt1 exist in PAH tissues and hypoxia-mediated HPASMCs. Cell proliferation, apoptosis and differentiation detection showed that miR-140-5p inhibits proliferation and promotes apoptosis and differentiation of HPASMCs in hypoxia, while the effect of Dnmt1 on hypoxia-mediated HPASMCs is reversed. Luciferase assay confirmed that miR-140-5p targets Dnmt1 directly. An inverse correlation is also found between miR-140-5p and Dnmt1 in HPASMCs. In addition, we further investigated whether miR-140-5p and Dnmt1 regulate HPASMC proliferation, apoptosis and differentiation by regulating SOD2 expression, and the results confirmed our speculation. Taken together, these results indicated that miR-140-5p at least partly targets Dnmt1 and regulates SOD2 expression to inhibit proliferation and promote apoptosis and differentiation of HPASMCs in hypoxia.
微小RNA-140-5p（miR-140-5p）在肺动脉高压（PAH）患者及PAH实验模型中表达下调，且在体外可抑制缺氧介导的肺动脉平滑肌细胞（PASMC）增殖。给予合成的miR-140-5p可预防和治疗已形成的实验性PAH。DNA甲基转移酶1（Dnmt1）在PAH相关的人肺动脉平滑肌细胞（HPASMC）中表达上调，其通过超氧化物歧化酶2（SOD2）启动子内CpG岛的高甲基化及下调SOD2表达来促进PAH的发展。我们使用TargetScan、PicTar和MiRanda工具搜索miR-140-5p的靶标，发现Dnmt1是miR-140-5p的潜在靶标。基于这些发现，我们推测miR-140-5p可能靶向Dnmt1并调节SOD2表达，从而调控缺氧介导的HPASMC增殖、凋亡和分化。我们分别通过定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测miR-140-5p、Dnmt1和SOD2的表达，发现PAH组织及缺氧介导的HPASMC中miR-140-5p和SOD2表达下调，Dnmt1表达上调。细胞增殖、凋亡和分化检测表明，miR-140-5p在缺氧状态下可抑制HPASMC增殖并促进其凋亡和分化，而Dnmt1对缺氧介导的HPASMC的影响则相反。荧光素酶报告基因检测证实miR-140-5p可直接靶向Dnmt1。在HPASMC中还发现miR-140-5p与Dnmt1呈负相关。此外，我们进一步研究了miR-140-5p和Dnmt1是否通过调节SOD2表达来调控HPASMC增殖、凋亡和分化，结果证实了我们的推测。综上所述，这些结果表明miR-140-5p至少部分靶向Dnmt1并调节SOD2表达，以抑制缺氧状态下HPASMC的增殖并促进其凋亡和分化。
