【摘要】 目的:探讨microRNA-25(miR-25)对缺氧/复氧诱导的H9C2细胞纤维化的作用及机制。方法:建立miR-25过表达/沉默的H9C2细胞株,行缺氧/复氧损伤处理,Real-time PCR检测miR-25的表达,Western blot检测TIMP2、MMP2、CollagenⅠ、CollagenⅢ、Fibronectin及HMGB1蛋白表达水平。构建转染HMGB1 shRNA的H9C2稳定细胞系,行缺氧/复氧损伤处理,Western blot检测HMGB1、TIMP2、MMP2、CollagenⅠ、CollagenⅢ蛋白表达水平。双荧光素酶试验验证miR-25与HMGB1的靶向关系。结果:miR-25高表达组,HMGB1表达减少,TIMP2、MMP2、CollagenⅠ、CollagenⅢ、Fibronectin蛋白表达减少,细胞纤维化程度降低,与对照组相比,差异具有统计学意义(P<0. 01)。转染HMGB1-shRNA组H9C2细胞,缺氧/复氧处理后,HMGB1、TIMP2、MMP2、CollagenⅠ、CollagenⅢ蛋白表达减少,与对照组相比,差异具有统计学意义(P<0. 01)。双荧光素报告基因显示,转染miR-25mimic+野生型HMGB1-3’UTR报告基因载体组荧光素酶信号强度明显下降;对照组荧光素酶没有变化,差异具有统计学意义(P<0. 05)。结论:miR-25通过靶向调控HMGB1表达降低缺氧/复氧诱导的H9C2细胞纤维化。更多还原

【Abstract】 Objective: To investigate the effect and mechanism of miR-25 on myocardial fibrosis induced by hypoxia/reoxygenationin H9C2 cell. Methods: The H9C2 cells with over-expression of miR-25 were treated with hypoxia/reoxygenation,Real-time PCR was used to detect the expression of miR-25,Western blot was performed to examine the protein-level expression of TIMP2,MMP2,CollagenⅠ,CollagenⅢ,Fibronectin and HMGB1. The H9C2 cells transfected with HMGB1-shRNA were subjected to hypoxia/reoxygenation,Western blot was performed to examine the protein-level expression of TIMP2,MMP2,CollagenⅠ,CollagenⅢ and HMGB1. Dualluciferase reporter assay was used to confirm that HMGB1 was the target gene of miR-25 in the H9C2 cells. Results: Over-expression of miR-25 significantly reduced the protein-level expression of HMGB1,TIMP2,MMP2,Collagen Ⅰ,Collagen Ⅲ and Fibronectin H9C2 cell in duced by hypoxia/reoxygenation(P<0. 05). The H9C2 cells transfected with HMGB1-shRNA was treated by hypoxia/reoxygenation,the expression of HMGB1,TIMP2,MMP2,Collagen Ⅰ,Collagen Ⅲ were down-regulated(P<0. 05). The result of dual-luciferase reporter assay showed that compared with control group,transfection with HMGB1-3’ UTR-psi-CHECK2 + miR-25 mimic strongly inhibited luciferase activity(P < 0. 05). Conclusion: microRNA-25 protect against myocardial fibrosis in H9C2 cell induced by hypoxia/reoxygenation through HMGB1 pathway.更多还原