miR-760影响充血性肺动脉高压肺动脉肌化改变的机制研究

Study on the mechanism of miR-760 in affecting pulmonary arterial muscularization in patients with congestive pulmonary arterial hypertension

目的:探讨微小RNA-760(miR-760)通过调控toll样受体4(TLR4)引起充血性肺动脉高压肺动脉肌化的机制。方法:使用实时定量PCR法检测猪肺动脉平滑肌细胞(PASMCs)和氨基马尿酸处理的PASMCs中miR-760的表达;采用病毒包装载体构建miRNA-760过表达和沉默载体,分别检测对肺动脉平滑肌细胞增殖和下游血小板TLR4蛋白表达的影响。使用双荧光素酶报告基因实验检测miR-760和TLR4基因的靶向性。结果:cPAH-PASMCs组miR-760的表达水平显著低于PASMCs组;外源性上调miR-760可有效抑制TLR4蛋白表达、控制PASMCs增殖;双荧光素酶报告实验显示野生型TLR4基因共转染miR-760过表达载体后荧光素酶活性较PASMCs组显著降低。结论:miR-760可能通过靶向TLR4影响充血性肺动脉高压肺动脉肌化的改变,miR-760可能是充血性肺动脉高压发病的保护因素。

Objective: To investigate the mechanism of pulmonary arterial muscularization in congestive pulmonary hypertension induced by microRNA-760(miR-760) through regulating Toll-like receptor 4(TLR4). Methods: Real-time quantitative polymerase chain reaction was used to detect the expression of miR-760 of porcine pulmonary artery smooth muscle cells(PASMCs) in control group and in aminohippuric acid-treated PASMCs(cPAH-PASMCs) group. The viral packaging vectors were used to construct the overexpression and silencing vectors of miR-760. The effects of miR-760 on the proliferation of PASMCs and the expression of TLR4 protein in downstream platelets were detected. The dual luciferase reporter gene assay was used to detect the targeting of miR-760 and TLR4 genes. Results: The expression level of miR-760 in cPAH-PASMCs group was significantly lower than that in the PASMCs group. Exogenous up-regulation of miR-760 could effectively inhibit the expression of TLR4 protein and control the proliferation of PASMCs. The dual luciferase reporter assay showed that after the wild-type TLR4 gene was cotransfected into the miR-760 overexpression vector,the luciferase activity was significantly lower than that of PASMCs group. Conclusion: miR-760 may affect pulmonary artery muscularization in patients with congestive pulmonary hypertension by targeting TLR4. miR-760 may be a protective factor for the pathogenesis of cPAHs.