Background

Bicuspid aortic valve is a heritable heart valve disease with characteristic early onset of valve calcification and stenosis with rapid progression compared with tricuspid aortic valve. Strong evidence indicates that many miRNAs and their target genes are involved in the development of BAV calcification. This study was designed to investigate miR-15a and Smad7 expressions in BAV and their mechanism of regulating calcification.

Methods

Immunohistochemistry and western blotting were used to explore the expression levels of Smad7 and Runx2 in human aortic valve tissue. The expression of miR-15a, Smad7 and Runx2 were detected by RT-PCR. The relationship between miR-15a and Smad7 was assessed by dual-luciferase assay. MiR-15a was overexpressed in cultured porcine valve interstitial cells (PVICs) and the variations of Smad7 and Runx2 mRNA were evaluated by RT-PCR, as well as the changes of Smad7 and Runx2 protein. Alizarin reds staining was used to verify the calcification inhibition effect of miR-15a in cultured PVICs under calcification induction.

Results

Compared with calcified TAV, remarkable higher expression of Smad7 and Runx2 were found in calcified BAV, significantly lower expressions of miR-15a and higher expressions of Smad7 and Runx2 mRNA, along with negative correlation between expressions of miR-15a and mRNA level of Smad7. MiR-15a-overexpressed PVICs shown upregulation of miR-15a, downregulation of Smad7 and Runx2 expression. PVICs under calcification induction shown significantly reduced calcification in miR-15a-overexpressed group.

Conclusions

The lower miR-15a expression in BAV results in the high expression of Smad7, thereby reducing antagonism of Smad2/3–Smad4 complex to runx2 and promoting the progress of BAV calcification.

中文翻译：

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MiR-15a通过抑制Smad7通过TGF-β信号通路调节二尖瓣主动脉钙化

背景

二尖瓣主动脉瓣膜炎是一种遗传性心脏瓣膜疾病，与三尖瓣主动脉瓣膜相比，特征在于瓣膜钙化和狭窄的早期发作且进展迅速。有力的证据表明，许多miRNA及其靶基因都参与了BAV钙化的发展。本研究旨在研究BAV中miR-15a和Smad7的表达及其调节钙化的机制。

方法

免疫组化和免疫印迹用于探讨Smad7和Runx2在人主动脉瓣组织中的表达水平。RT-PCR检测miR-15a，Smad7和Runx2的表达。通过双荧光素酶分析评估了miR-15a和Smad7之间的关系。MiR-15a在培养的猪瓣膜间质细胞（PVIC）中过表达，并通过RT-PCR评估Smad7和Runx2 mRNA的变化，以及Smad7和Runx2蛋白的变化。茜素红染色用于验证钙诱导下培养的PVIC中miR-15a的钙化抑制作用。

结果

与钙化TAV相比，钙化BAV中Smad7和Runx2的表达明显升高，miR-15a的表达显着降低，Smad7和Runx2的mRNA表达升高，miR-15a的表达与Smad7的mRNA水平呈负相关。MiR-15a过表达的PVIC显示miR-15a上调，Smad7和Runx2表达下调。钙化诱导下的PVIC在miR-15a过表达组中显示钙化明显降低。

结论

BAV中较低的miR-15a表达导致Smad7的高表达，从而降低Smad2 / 3–Smad4复合物对runx2的拮抗作用并促进BAV钙化的进程。