二叶式主动脉瓣中miRNA-195调控瓣膜钙化的机制研究

目的：探究二叶式主动脉瓣中miRNA-195调控瓣膜钙化的可能机制。方法：收集35例接受主动脉瓣置换术患者狭窄的二叶式或三叶式主动脉瓣，通过PCR及Western blot分别检测miRNA-195的表达量、果蝇母源抗皮肤生长因子（drosophila mothers against decapentaplegic 7，SMAD7）的mRNA及蛋白表达量。双荧光素酶法预测miRNA-195的靶基因。在猪瓣膜间质细胞中分别沉默和过表达miRNA-195，检测SMAD7的mRNA表达水平并探究两者的相互关系。在人类瓣膜组织上通过免疫组化染色检测基质金属蛋白酶（matrix metalloproteinase，MMP）-2、MMP-9蛋白水平，通过天狼星红染色检测胶原蛋白水平，通过Von Kossa染色比较钙化程度差异，进而研究钙化相关的功能改变。结果：与狭窄的三叶式主动脉瓣相比，狭窄的二叶式主动脉瓣中miRNA-195表达降低，SMAD7的mRNA和蛋白水平升高。双荧光素酶实验提示SMAD7是miRNA-195的直接靶标。在猪瓣膜间质细胞中沉默miRNA-195引起SMAD7 mRNA水平升高，过表达miRNA-195引起SMAD7 mRNA水平降低。狭窄钙化的二叶式主动脉瓣组织中MMP-2、MMP-9及胶原的表达较狭窄钙化的三叶式主动脉瓣升高。结论：在二叶式主动脉瓣中，下调的miRNA-195促进了SMAD7表达，进而促进细胞外基质的纤维化并最终促进其钙化。

Calcification mechanism regulated by miRNA-195 in bicuspid aortic valve

Objective: To investigate the calcification mechanism of bicuspid aortic valve(BAV) which involves the microRNA-195. Methods: Stenotic BAV or degenerative tricuspid aortic valve(DTV) samples were collected from 35 patients undergoing aortic valve replacement. The mRNA expression of miRNA-195 and SMAD7, and protein expression of SMAD7 were determined by RT-PCR and Western blot, respectively. Dual-luciferase assay was performed to determine putative target of miRNA-195. In porcine valve interstitial cells(VIC) ,we down-regulated and up-regulated the microRNA-195 level, and examine the mRNA expression of SMAD7 to explore the relationship between miRNA-195 and SMAD7. Moreover, in human leaflet samples, immunochemistry staining was carried out to test the protein expression of MMP-2 and MMP-9, and Sirus Red staining was done to investigate the expression of collagen, and the Von Kossa staining was implemented to explore the mineralization nodes. All of these were done to compare the functional alteration associated with cellular calcification. Results: Compared with TAV, the expression of miRNA-195 was remarkably lower in BAV with higher expression of SMAD7. Luciferase experiments validated that miRNA-195 directly targeted SMAD7. Overexpression of miRNA-195 inhibited the mRNA expression of SMAD7, while miRNA-195 inhibitor treatment resulted in increased SMAD7 expression in VIC. Finally, more MMP-2 and MMP-9 expression and more collagen distribution were observed in BAV than in DTV. Conclusion: Our study has demonstrated that miRNA-195 is much more down-regulated in stenotic BAV than that in DTV. The downregulation of miRNA-195 promotes valvular calcification via targeting SMAD7, which involves the fibrosis of extracellular matrix.