MiR-761 promotes aortic valve calcification by inhibiting GABARAP expression

To investigate the effect and potential mechanism of miR-761 on calcified aortic valve disease（CAVD）.

Normal（n=6）and calcified（n=8）valve tissues were collected from Department of Cardiothoracic Surgery of the First Affiliated Hospital of Chongqing Medical University between January 2022 and January 2023. The changes of autophagy, osteogenesis, GABARAP protein or miR-761 expression in calcified valve tissues were detected by Western blotting, immunohistochemical staining and RT-qPCR. Double luciferase assay verified miR-761 binding to GABARAP 3′-UTR region. The phenotypes of porcine aortic valve interstitial cells（pVICs）were identified by immunofluorescence assay. pVICs were transfected with miR-761 mimic, inhibitor and corresponding control to construct pVICs cell lines with over-expression or knockdown of miR-761. After adding GFP-LC3 adenovirus, the number of autophagosomes was observed. Western blotting, alkaline phosphatase staining and alizarin red S staining were used to detect the osteogenic differentiation of pVICs before and after osteogenic medium（OM）treatment.

Compared with normal tissues, the prdein levels of Beclin 1, ULK1, α-SMA, RUNX2 and GABARAP were up-regulated in calcified valve tissue tissues, while the levels of p62 and miR-761 were down-regulated（P<0.05）. Dual luciferase assay verified the binding of miR-761 to GABARAP 3′-UTR region（P<0.05）. The isolated pVICs expressed interstitial cell markers（α-SMA, Vimentin）. Confocal microscopy showed that overexpression of miR-761 reduced autophagosome production in pVICs. The protein results, alkaline phosphatase staining and alizarin red S staining showed that knockdown of miR-761 significantly increased the expression of GABARAP in pVICs, significantly decreased the expressions of OPN, α-SMA, CollagenⅠand RUNX2（P<0.05）, and inhibited the calcium salt deposition in the cells induced by OM.

MiR-761 may inhibit autophagy and promote cardiac valve calcification by inhibiting GABARAP expression.