miR-323通过FGF9调控缺氧诱导的心肌H9C2细胞凋亡

目的:探讨微小RNA-323(miR-323)对缺氧诱导大鼠心肌H9C2细胞凋亡的影响及其机制。方法:建立缺氧损伤的H9C2细胞模型,在H9C2细胞转染anti-miR-323、pcDNA-FGF9和si-FGF9,并缺氧处理48 h。采用qPCR检测miR-323的表达, Western blot检测成纤维细胞生长因子9(FGF9)、cleaved caspase-3、c-Jun氨基末端激酶(JNK)和p-JNK的蛋白水平,MTT比色法检测细胞活力,流式细胞术检测细胞凋亡。生物学信息预测miR-323的靶基因并用双萤光素酶报告基因进一步验证。结果:缺氧明显降低H9C2细胞12 h、24 h和48 h的细胞活力(P<0.05),显著提高细胞凋亡率和cleaved caspase-3的蛋白水平(P<0.05),效应均呈时间依赖性。缺氧处理的H9C2细胞中miR-323和p-JNK水平上调,FGF9蛋白表达下调(P<0.05)。下调miR-323表达或过表达FGF9显著提高缺氧处理的H9C2细胞活力,降低细胞凋亡率和cleaved caspase-3蛋白的水平(P<...

miR-323 regulates hypoxia-induced apoptosis of H9C2 cardiomyocytes through FGF9

AIM:To investigate the effect of microRNA-323(miR-323)on the apoptosis of hypoxia-induced rat H9C2 cardiomyocytes and its mechanism.METHODS:The hypoxic injury model was established in the H9C2 cells.Anti-miR-323,pcDNA-FGF9 and si-FGF9 were transfected into the H9C2 cells and cultured under hypoxic condition for 48 h.The expression of miR-323 was detected by qPCR.The protein levels of fibroblast growth factor 9(FGF9),cleaved caspase-3,c-Jun N-terminal kinase(JNK)and p-JNK were determined by Western blot.The cell viability was measured by MTT assay,and the apoptosis was analyzed by flow cytometry.The method of bioinformatics was applied to predict the target gene of miR-323,and dual-luciferase reporter assay was used for further validation.RESULTS:Hypoxia greatly reduced the viability of H9C2 cells at 12 h,24 h and 48 h(P<0.05),and remarkably increased apoptotic rate and the protein level of cleaved caspase-3 in a time-dependent manner(P<0.05).The expression of miR-323 and the protein level of p-JNK were up-regulated and the expression of FGF9 was down-regulated in the H9C2 cells exposed to hypoxia(P<0.05).Down-regulation of miR-323 and over-expression of FGF9 obviously increased the viability of the H9C2 cells exposed to hypoxia,and decreased the apoptotic rate and the protein level of cleaved caspase-3(P<0.05).FGF9 was the target gene of miR-323.Down-regulation of FGF9 reversed the attenuating effect of down-regulation of miR-323 on hypoxia-induced H9C2 cell injury.miR-323 regulated FGF9 and affected p-JNK level.CONCLUSION:miR-323 affects the viability and apoptosis of H9C2 cardiomyocytes under hypoxia by targeting FGF9 and regulating JNK signaling pathway.