Upregulated microRNA-330-3p promotes calcification in the bicuspid aortic valve via targeting CREBBP

One key risk factor of aortic valve stenosis in clinical practice is bicuspid aortic valve (BAV). Increasing evidence indicates that numerous microRNAs (miRs/miRNAs) are involved in BAV calcification via their target genes. miR-330-3p was found to be involved in the deterioration of BAV calcification by miR profiling in human calcified BAV and tricuspid aortic valve (TAV) tissues in the present study and the underlying mechanism was investigated. RNA sequencing was performed on four BAV and four TAV tissues from patients with aortic stenosis before these leaflets were examined for the expression levels of miR-330-3p and CREB-binding protein (CREBBP) by reverse transcription-PCR. The alteration of functional factors associated with calcification was also assessed by Western blotting and immunohistochemistry in human aortic tissue samples. The putative target of miR-330-3p was detected by dual-luciferase assay in 293 cells. Furthermore, the influence of miR-330-3p expression on osteogenic progression was explored in cultured porcine valve interstitial cells (VICs). Rescue experiments of CRBBP were performed to confirm the influence of the miR-330-3p-CREBBP pathway in the calcification progress in porcine VICs. RNA sequencing indicated distinct expression of miR-330-3p in human BAV tissues compared with TAV, which was then confirmed by PCR. CREBBP expression levels in human BAV and TAV leaflets also demonstrated the opposite alterations. This negative correlation was then confirmed in cultured porcine VICs. Under an osteogenic environment, cellular calcification was promoted in miR-330-3p-overexpressed porcine VICs expressing higher bone morphogenetic protein 2, Runt-related transcription factor 2, matrix metalloproteinase (MMP)-2, MMP-9 and collagen I compared with controls. Rescue experiments further confirmed that miR-330-3p played its role via targeting CREBBP in porcine VICs. Collectively, miR-330-3p was upregulated in calcified BAV compared with TAV. The upregulation of miR-330-3p promotes the calcification progress partially via targeting CREBBP.