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

目的 研究微小核糖核酸141（miRNA-141）调控骨形成蛋白-2（Bone morphogenetic protein 2, BMP-2）诱导人主动脉瓣钙化的机制。方法 收集24例人退行性主动脉瓣，用实时荧光定量聚合酶链式反应（RT-qPCR）及蛋白免疫印迹（Western Blot）检测miRNA-141和BMP-2 mRNA及蛋白的表达水平。人原代主动脉瓣膜间质细胞（human aortic valve interstitial cells, HAVIC）中上/下调miRNA-141表达，Von Kossa染色比较细胞钙化，并比较远端缺失同源盒5（Distal-less Homeobox 5, Dlx5）的mRNA以及BMP-2蛋白表达；双荧光素酶法验证Dlx5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，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HAVICs中，上/下调miRNA-141可抑制/促进钙化（P=0.02或P=0.01），并降低/升高Dlx5的mRNA表达（P均=0.01）以及BMP-2蛋白表达。双荧光素酶法验证Dlx5为miRNA-141的靶基因。上/下调主动脉瓣钙化小鼠miRNA-141可抑制/促进钙化（P<0.05），并降低/升高Dlx5及BMP-2表达（P<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基因调控BMP-2蛋白，诱导人主动脉瓣钙化。

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

Objective To investigate the mechanism of miRNA-141 regulating bone morphogenetic protein-2 (BMP-2)inhuman aortic valve calcification. Methods 24 degenerative aortic valve samples were collected, and the expression of miRNA-141 and BMP-2 were determined. In human aortic valve interstitial cells (HAVICs), miRNA-141 was down/up-regulated, and Von Kossa staining was implemented to show cellular calcification. The mRNA expression of Dlx5 and protein expression of BMP-2 were examined in HAVICs and luciferase experiments were used to detect the targeting relationship between miRNA-141 andDlx5.In aortic valve calcification mouse models with or without Dlx5 knockout, miRNA-141 was down/up-regulated, and Von Kossa staining was implemented to evaluate valvular calcification, and protein expression of BMP-2 was examined. Results Compared with normal valves, the expression of miRNA-141 was remarkably lower in degenerative aortic valves, and mRNA/protein expression of BMP-2 was significantly higher. In HAVICs, overexpression of miRNA-141attenuated calcification and was associated with decreasedDlx5/BMP-2 expression, while knockdown of miRNA-141 was on the contrary. Luciferase experiments validated that miRNA-141 directly targeted Dlx5. In aortic valve calcification mouse model, overexpression of miRNA-141 was also associated with decreased calcification and Dlx5/BMP-2 expression, while knockdown of miRNA-141 was on the contrary. In mouse model withDlx5 knockout, there were no correlation between miRNA-141 expression and valvular calcification, or BMP-2 expression.Conclusion miRNA-141 promotes human aortic valve calcification via regulating BMP-2 by targeting Dlx5.