摘要
目的:探讨微小RNA-25(miR-25)在缺氧/复氧诱导的H9c2细胞凋亡过程中的作用及机制。方法:构建miR-25高表达H9c2细胞系,行缺氧/复氧损伤处理,real-time PCR检测miR-25及高迁移率族蛋白B1(HMGB1)mRNA的表达,Western blot检测HMGB1及细胞凋亡相关蛋白Bcl-2和cleaved caspase-3的蛋白表达水平,流式细胞术检测细胞凋亡及细胞周期的变化,明确miR-25的高表达对缺氧/复氧所诱导的H9c2细胞凋亡及HMGB1表达的影响。然后通过双萤光素酶报告基因验证miR-25与HMGB1的靶向关系,并构建转染HMGB1 shRNA的H9c2稳定细胞系,行缺氧/复氧损伤处理,验证HMGB1是否参与缺氧/复氧诱导的H9c2细胞凋亡的调控。结果:与对照组相比,高表达miR-25组的H9c2细胞在缺氧/复氧诱导后HMGB1表达下调,Bcl-2表达上调,cleaved caspase-3蛋白水平下调,流式细胞术提示处于S期的细胞增多,G1期减少(P<0.01)。双萤光素报告基因显示,转染miR-25 mimic+野生型HMGB1-3’UTR报告基因载体组萤光素酶活性明显下降;转染miR-25 inhibitor+野生型HMGB1-3’UTR和转染miR-25 mimic+突变型HMGB1-3’UTR报告基因载体组萤光素酶活性没有变化。转染HMGB1-shRNA的H9c2细胞中抗凋亡蛋白Bcl-2表达上调,凋亡蛋白cleaved caspase-3蛋白水平下调,流式细胞术分析提示细胞凋亡减少,处于S期的细胞增多,G1期减少(P<0.05)。结论:miR-25减少缺氧/复氧诱导的H9c2细胞凋亡,期作用机制可能与靶向调控HMGB1表达有关。
AIM： To investigate the role and mechanism of microRNA-25（ miR-25） in apoptosis of H9 c2 cells induced by hypoxia/reoxygenation. METHODS： The H9 c2 cells with over-expression of miR-25 were treated with hypoxia/reoxygenation. Real-time PCR was used to detect the expression of miR-25 and high mobility group box-1（ HMGB1）mRNA. Western blot was performed to examine the protein expression levels of HMGB1,Bcl-2 and cleaved caspase-3.Flow cytometry was used to analyze the proportion of apoptotic cells and the cell cycle. Dual-luciferase reporter assay was used to confirm that HMGB1 was the target gene of miR-25 in the H9 c2 cells. Moreover,the H9 c2 cells transfected with HMGB1-shRNA were subjected to hypoxia/reoxygenation to verify whether HMGB1 participated in the regulation of apoptosis of H9 c2 cells. RESULTS： Over-expression of miR-25 significantly reduced the protein expression levels of HMGB1 and cleaved caspase-3,and increased the expression of Bcl-2 and the entrance into S phase in H9 c2 cells induced by hypoxia/reoxygenation（ P〈0. 01）. The result of dual-luciferase reporter assay showed that compared with the control group,transfection with HMGB1-3＇UTR-psi-CHECK2 ＋ miR-25 mimic strongly inhibited the luciferase activity（ P〈0. 05）. After the H9 c2 cells transfected with HMGB1-shRNA was treated with hypoxia/reoxygenation,the expression of Bcl-2 was upregulated,the expression of cleaved caspase-3 was down-regulated,and the cells in S phase were increased（ P〈0. 05）.CONCLUSION： miR-25 reduces apoptosis of H9 c2 cells induced by hypoxia/reoxygenation,and its mechanism may be related with the inhibition of HMGB1 expression via interacting with its 3＇-UTR.
作者
刘启方
黄晶
徐敏
LIU Qi-fang;HUANG Jing;XU Min(Department of Cardiology;Department of Rehabilitation,Guizhou Province People’s Hospital,Guiyang 550002,China.)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2018年第7期1214-1221,共8页
Chinese Journal of Pathophysiology
