Calcification Mechanism Involing MicroRNA-195 In Bicuspid Aortic Valve

Objective:The progression of aortic stenosis caused by leaflet calcification in bicuspid aortic valve(BAV)patients is more accelerative compared to patients with trileaflet aortic valve(TAV)although the underlying mechanisms are still poorly defined.Micro-RNAs(miRNAs)are essential post-transcriptional modulators of gene expression and miRNA-195 was known to be downregulated in stenotic BAVs compared with insufficient BAVs.We hypothesized that the dysregulation of miRNA-195 in BAV promotes the calcification of valve interstitial cells(VIC)via modulating SMAD7,a calcification-related target gene of miR-195,which causes us to carry out experiments to test this hypothese.Methods:Stenotic aortic valve samples(BAV or TAV)were collected from 50 patients undergoing aortic valve replacement.The mRNA expressions of miRNA-195 and SMAD7 were determined in BAV and TAV samples.Immunohistochemical analyses and western blot were carried out to check SMAD7 protein expression in human samples.The dual-luciferase reporter assay was performed to determine putative target of miR-195.Porcine VICs were transfected with miRNA-195 mimic or miRNA-195 inhibitor to further explore the relationship between miRNA-195 and SMAD7.Moreover,the alteration associated cellular calcification(the expression of Runx2,the degree of calcium deposit,the levels of matrix metalloprotein-2,matrix metalloprotein-9,and collagen)were compared in all human samples.or in the cells insufficient of miR-195.Results:compared with TAV leaflets,the expression of miRNA-195 was remarkably lower in BAV leaflets associating with higher mRNA and protein expression of SMAD7.Luciferase experiments validated that miRNA-195 directly target SMAD7,Overexpression of miR-195 in porcine VICs inhibited the mRNA expression of SMAD7 while miR-195 inhibitor treatment resulted in increased SMAD7 mRNA expression.When it comes to the functional alteration,we observed more severe calcium deposit,more Runx2 expression and MMP2 expression in the cells insufficient of miR-195.We observed more MMP2 and MMP9 expression,and more collagen distribution in human calBAV leaflets compared with the TAV leaflets.Conclusions:Our study has demonstrated that miRNA-195 is downregulated in stenotic aortic leaflets from BAV patients compared with patients with TAV.We have also shown that downregulation of miR-195 promotes valvular calcification via targeting SMAD7,which involves the fibrosis of extracellular matrix in BAV leaflets.Our findings provide new clues regarding the mechanism of accelerated valvular calcification in BAV patients.