miR-181d-5p promotes valvular calcification and directly targets Wnt inhibitory factor 1 in calcific aortic valve disease.

This study aimed to investigate the role of miR-181d-5p in the pathogenesis of calcific aortic valve disease (CAVD), identify its downstream targets, and provide novel insights into therapeutic strategies for this condition. The differential expression of miRNAs in valve samples from CAVD patients was assessed via qRT-PCR to identify key regulatory miRNAs. Target gene prediction software and four Gene Expression Omnibus (GEO) datasets (GSE12644, GSE235995, GSE51472, and GSE77287) were employed to identify differentially expressed genes (DEGs) in CAVD and predict the downstream targets of miR-181d-5p. An in vitro calcification model was established using human aortic valvular interstitial cells (hAVICs), which were transfected with a miR-181d-5p mimic, an inhibitor, or their respective negative controls to evaluate the effect of miR-181d-5p on calcification. The direct binding between miR-181d-5p and its target gene was validated using a dual-luciferase reporter assay. qRT-PCR revealed that the expression levels of miR-181d-5p, miR-181c-5p, and miR-31-5p were significantly increased in CAVD valve tissues compared with those in the non-calcified aortic regurgitation group (P < 0.01). Furthermore, plasma levels of miR-181d-5p, miR-181c-5p, miR-31-5p, and miR-19b-3p were significantly elevated in patients with calcification (both with and without stenosis) compared to healthy controls (P < 0.05). Bioinformatics analysis and target prediction tools identified Wnt inhibitory factor 1 (WIF1) as a potential target of miR-181d-5p. qRT-PCR confirmed that WIF1 expression was significantly downregulated in CAVD valves (P < 0.05). In vitro assays demonstrated that miR-181d-5p expression was upregulated in the calcification model compared to the blank control (P < 0.05). Western blot analysis indicated that on day 7 of osteogenic induction, the protein levels of the osteogenic markers RUNX2 and OPN were increased, whereas WIF1 protein expression was decreased. Following osteogenic induction, Alizarin Red S staining revealed that transfection with the miR-181d-5p mimic exacerbated calcium nodule formation compared to the control, whereas transfection with the miR-181d-5p inhibitor significantly attenuated this effect. The dual-luciferase reporter assay confirmed a direct targeting relationship between miR-181d-5p and WIF1. In conclusion, miR-181d-5p promotes valvular calcification, possibly by directly targeting and inhibiting WIF1, suggesting its potential as a novel therapeutic target for CAVD.