微RNA-141靶向Dlx5基因对骨形态发生蛋白2诱导人主动脉瓣钙化的调控作用  

Regulatory effect of microRNA-141 targeting Dlx5 on bone morphogenetic protein-2 induced-calcification of human aortic valve

作　　者：杨潜[1] 王俊男 凌新宇 薛小飞 肖健[1] 奚望[1] 王志农[1] YANG Qian;WANG Jun-nan;LING Xin-yu;XUE Xiao-fei;XIAO Jian;XI Wang;WANG Zhi-nong(Department of Cardiothoracic Surgery,Changzheng Hospital,Naval Medical University(Second Military Medical University),Shanghai 200003,China)

机构地区：[1]海军军医大学(第二军医大学)长征医院胸心外科,上海200003

出　　处：《第二军医大学学报》2020年第12期1309-1313,共5页Academic Journal of Second Military Medical University

基　　金：上海市卫生和计划生育委员会科研课题(201640172);上海市领军人才计划(2015044)。

摘　　要：目的探讨miRNA-141对骨形态发生蛋白2(BMP-2)诱导人主动脉瓣钙化的调控作用及机制。方法收集24例人退行性主动脉瓣,用qRT-PCR及蛋白质印迹法检测miRNA-141和BMP-2的mRNA及蛋白表达水平。在人主动脉瓣膜间质细胞(HAVIC)中上/下调miRNA-141表达,通过Von Kossa染色比较细胞钙化,并比较远端缺失同源盒5(Dlx5)mRNA和BMP-2蛋白表达;双荧光素酶报告基因实验验证Dlx5是否为miRNA-141的靶基因。在主动脉瓣钙化小鼠和Dlx5基因敲除主动脉瓣钙化小鼠中,上/下调miRNA-141表达,通过Von Kossa染色比较主动脉瓣钙化,并检测BMP-2蛋白表达。结果与正常主动脉瓣膜组织相比,人退行性主动脉瓣miRNA-141的表达降低(1.00±0.02 vs 0.35±0.06,P=0.01),BMP-2的mRNA及蛋白表达增加(P均=0.01)。在HAVIC中,上调/下调miRNA-141可抑制/促进钙化(P=0.02或P=0.01),并降低/升高Dlx5 mRNA表达(P均=0.01)及BMP-2蛋白的表达(P=0.02或P=0.01)。双荧光素酶报告基因实验验证了Dlx5为miRNA-141的靶基因。上调/下调主动脉瓣钙化小鼠miRNA-141可抑制/促进钙化(P均=0.01),并降低/升高Dlx5、BMP-2 mRNA及蛋白的表达(P均<0.05);Dlx5基因敲除小鼠中,上/下调miRNA-141不影响瓣膜钙化和BMP-2的表达。结论miRNA-141靶向Dlx5基因抑制BMP-2蛋白诱导的人主动脉瓣钙化。Objective To investigate the regulating effects and mechanism of microRNA(miRNA)-141 on bone morphogenetic protein-2(BMP-2)-induced calcification of human aortic valve.Methods Twenty-four samples of human degenerative aortic valve were collected,and the mRNA and protein expression levels of miRNA-141 and BMP-2 were detected by quantitative real-time polymerase chain reaction and Western blotting.miRNA-141 was up/down-regulated in human aortic valve interstitial cells(HAVICs),Von Kossa staining was used to show cellular calcification,and mRNA expression of distal-less homeobox 5(Dlx5)and protein expression of BMP-2 were compared.Dual luciferase experiment was used to verify whether Dlx5 was the target gene of miRNA-141.miRNA-141 was up/down-regulated in aortic valve calcification mouse models with or without Dlx5 knockout,Von Kossa staining was used to compare aortic valve calcification,and BMP-2 protein expression was detected.Results Compared with normal aortic valve tissues,the expression of miRNA-141 was significantly decreased in degenerative aortic valves(1.00±0.02 vs 0.35±0.06,P=0.01),while the mRNA and protein expression levels of BMP-2 were significantly increased(both P=0.01).In HAVICs,the up/down regulation of miRNA-141 could inhibit/promote calcification(P=0.02 or P=0.01),and decrease/increase the mRNA expression of Dlx5(both P=0.01)and the protein expression of BMP-2(P=0.02 or P=0.01).Dual luciferase experiment validated that miRNA-141 directly targeted Dlx5.In aortic valve calcification mouse model,up/down-regulation of miRNA-141 could inhibit/promote calcification(both P<0.05),and decrease/increase the mRNA and protein expression levels of Dlx5 and BMP-2(all P<0.05);while in mouse model with Dlx5 knockout,there were no correlation between miRNA-141 expression and valvular calcification or BMP-2 expression.Conclusion miRNA-141 can inhibit human aortic valve calcification via regulating BMP-2 by targeting Dlx5.

关 键 词：主动脉瓣 瓣膜钙化 微RNA-141 远端缺失同源盒5 骨形态发生蛋白2 

分 类 号：R737.11[医药卫生—肿瘤]