肺动脉高压(PAH)是一种以肺血管重构为特征的进行性疾病,这可能会导致右心衰竭甚至死亡。越来越多的证据证实microRNA-26家族在心血管疾病中起着关键作用;然而,它们在PAH中的功能仍然未知。这里,我们使用定量RT-PCR研究了miR-26家族在PAH患者血浆中的表达,并确定miR-26a-5p是最下调的成员,在低氧诱导的肺动脉平滑肌细胞(PASMC)模型和PAH患者的肺组织中自噬也降低。此外,染色质免疫沉淀(ChIP)分析和荧光素酶报告基因分析显示,缺氧诱导因子1α(HIF-1α)与miR-26a-5p的启动子特异性相互作用,并抑制其在PASMC中的表达。串联mRFP-GFP-LC3B荧光显微镜显示miR-26a-5p抑制缺氧诱导的PAMSC自噬,其特征是自噬体和自溶酶体的形成减少。此外,结果显示miR-26a-5p过表达能有效抑制PASMC的增殖和迁移,通过细胞计数试剂盒-8,EdU染色,伤口愈合,和transwell分析。机械上,PFKFB3,ULK1和ULK2是miR-26a-5p的直接靶标,如通过双荧光素酶报告基因测定和蛋白质印迹确定的。同时,PFKFB3可以进一步增强ULK1的磷酸化水平,促进PASMCs的自噬。此外,气管内给予adeno-miR-26a-5p可显着减轻缺氧诱导的PAH大鼠模型体内右心室肥厚和肺血管重塑。一起来看,HIF-1α/miR-26a-5p/PFKFB3/ULK1/2轴在低氧诱导的PASMC自噬和增殖的调控中起关键作用。MiR-26a-5p可作为诊断和治疗PAH的有吸引力的生物标志物。
Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease characterized by pulmonary vascular remodeling, which may cause right heart failure and even death. Accumulated evidence confirmed that microRNA-26 family play critical roles in cardiovascular disease; however, their function in PAH remains largely unknown. Here, we investigated the expression of miR-26 family in plasma from PAH patients using quantitative RT-PCR, and identified miR-26a-5p as the most downregulated member, which was also decreased in hypoxia-induced pulmonary arterial smooth muscle cell (PASMC) autophagy models and lung tissues of PAH patients. Furthermore, chromatin immunoprecipitation (ChIP) analysis and luciferase reporter assays revealed that hypoxia-inducible factor 1α (HIF-1α) specifically interacted with the promoter of miR-26a-5p and inhibited its expression in PASMCs. Tandem mRFP-GFP-LC3B fluorescence microscopy demonstrated that miR-26a-5p inhibited hypoxia-induced PAMSC autophagy, characterized by reduced formation of autophagosomes and autolysosomes. In addition, results showed that miR-26a-5p overexpression potently inhibited PASMC proliferation and migration, as determined by cell counting kit-8, EdU staining, wound-healing, and transwell assays. Mechanistically, PFKFB3, ULK1, and ULK2 were direct targets of miR-26a-5p, as determined by dual-luciferase reporter gene assays and western blots. Meanwhile, PFKFB3 could further enhance the phosphorylation level of ULK1 and promote autophagy in PASMCs. Moreover, intratracheal administration of adeno-miR-26a-5p markedly alleviated right ventricular hypertrophy and pulmonary vascular remodeling in hypoxia-induced PAH rat models in vivo. Taken together, the HIF-1α/miR-26a-5p/PFKFB3/ULK1/2 axis plays critical roles in the regulation of hypoxia-induced PASMC autophagy and proliferation. MiR-26a-5p may represent as an attractive biomarker for the diagnosis and treatment of PAH.