| 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。 |
| 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 |
| DOI：10.16781/j.0258-879x.2020.12.1309 |
| 投稿时间：2020-07-04修订日期：2020-12-08 |
| 基金项目: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）. |
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| Regulatory effect of microRNA-141 targeting Dlx5 on bone morphogenetic protein-2 induced-calcification of human aortic valve |
| 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
△Co-first authors.
* Corresponding author) |
| Abstract: |
| 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. |
| Key words: aortic valve valvular calcification micro RNA-141 distal-less homeobox 5 bon morphogenetic protein-2 |
