MiR-499靶向HMGB1保护缺氧复氧心肌细胞损伤的分子机制探讨  

Protective effect of microRNA-499 targeting high mobility group Box-1 protein on hypoxia/reoxygenation cardiomyocyte injury: a study on molecular mechanism

作　　者：周慧良 甘受益[1] 李宾[1] 冯光瑞 宋巧巧[2] 王淼[1] Zhou Huiliang;Gan Shouyi;Li Bin;Feng Guangrui;Song Qiaoqiao;Wang Miao(Department of Cardiovascular Medicine,Central Hospital of Xianning City,First Affiliated Hospital of Hubei University of Science and Technology,Hubei Province,Xianning 437000,China;不详)

机构地区：[1]咸宁市中心医院湖北科技学院附属第一医院心血管内科,咸宁437000 [2]湖北科技学院内科教研室,咸宁437000

出　　处：《中国循证心血管医学杂志》2019年第12期1456-1460,共5页Chinese Journal of Evidence-Based Cardiovascular Medicine

基　　金：十二五国家科技支撑计划(2011BAI11B01);咸宁市中心医院湖北科技学院附属第一医院项目资助(2018XYA006)

摘　　要：目的研究miR-499对缺氧复氧心肌细胞H9c2损伤的影响,并探讨其作用机制。方法运用免疫印迹法(Western blot)检测缺氧复氧心肌细胞中高迁移率族蛋白1(HMGB1)、B细胞淋巴瘤/白血病-2(Bcl-2)、免抗人单克隆抗体(Bax)、caspase-3的蛋白表达;将H/R+miR-con组(转染miRcon)、H/R+miR-499组(转染miR-499 mimics)、miR-con组(转染miR-con)、miR-499组(转染miR-499 mimics)、anti-miR-con组(转染anti-miR-con)、anti-miR-499组(转染anti-miR-499)、H/R+si-con组(转染si-con)、H/R+si-HMGB1组(转染si-HMGB1)、H/R+miR-499+Ctrl组(miR-499 mimics和pcDNA3.1共转染)、H/R+miR-499+HMGB1组(miR-499 mimics和pcDNA3.1-HMGB1共转染),均以脂质体法转染至H9c2细胞;实时荧光定量聚合酶链锁反应(qRT-PCR)检测各组细胞中miR-499的表达;流式细胞术检测各组细胞的凋亡;双荧光素酶报告基因检测实验检测各组细胞的荧光活性。结果与NC组相比,H/R组细胞中HMGB1蛋白表达显著升高,miR-499表达显著降低(P<0.05);过表达miR-499或敲减HMGB1均可显著下调H/R H9c2细胞凋亡率,显著下调Bax、caspase-3的蛋白表达,上调Bcl-2的蛋白表达(P均<0.05);过表达HMGB1可逆转miR-499对H/R H9c2细胞凋亡的抑制作用。结论MiR-499可抑制缺氧复氧心肌细胞H9c2凋亡,保护心肌细胞,其机制与靶向HMGB1有关,将可为心脏病的治疗提供新靶点。Objective To study the influence of microRNA-499(miR-499)on hypoxia/reoxygenation cardiomyocyte H9c2(H/R-H9c2)and discuss the effective mechanism.Methods The protein expressions of high mobility group Box-1 protein(HMGB1),Bcl-2,Bax and caspase-3 in H/R-H9c2 were detected by using Western blotting assay.H/R-H9c2 were transfected by using liposome method in H/R+miR-con group(transfected with miRcon),H/R+miR-499 group(transfected with miR-499 mimics),miR-con group(transfected with miR-con),miR-499 group(transfected with miR-499 mimics),anti-miR-con group(transfected with anti-miR-con),anti-miR-499 group(transfected with anti-miR-499),H/R+si-con group(transfected with si-con),H/R+si-HMGB1 group(transfected with si-HMGB1),H/R+miR-499+Ctrl group(co-transfected with miR-499 mimics and pcDNA3.1),and H/R+miR-499+HMGB1 group(co-transfected with miR-499 mimics and pcDNA3.1-HMGB1).The expression of miR-499 in H/R-H9c2 was detected by using real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)in all groups.The apoptosis of H/R-H9c2 was detected by using flow cytometry(FCM),and fluorescence activity of H/R-H9c2 was detected by using dual-luciferase reporter gene assay in all groups.Results Compared with NC group,the protein expression of HMGB1 increased significantly and expression of miR-499 decreased significantly in H/R group(P<0.05).The over-expression of miR-499 or knock-down of HMGB1 can significantly down-regulated H/R-H9c2 apoptosis rate and protein expressions of Bax and caspase-3,and up-regulated protein expression of Bcl-2(all P<0.05).The over-expression of HMGB1 could reverse the inhibitive effect of miR-499 on H/R-H9c2 apoptosis.Conclusion MiR-499 can inhibit the apoptosis of H/R-H9c2 and protect cardiomyocytes,and the mechanism is correlated to targeting HMGB1,which can provide a new target spot for heart disease treatment.

关 键 词：miR-499 高迁移率族蛋白1 缺氧复氧心肌细胞 凋亡 

分 类 号：R542.2[医药卫生—心血管疾病]