circ-CCND1 regulates the CCND1/P53/P21 pathway through sponging miR-138-5p in valve interstitial cells to aggravate aortic valve calcification

To discuss the effect and mechanism of circular-CCND1 (circ-CCND1) on the regulation of calcified aortic valve disease (CAVD). Differentially expressed circRNAs were screened through the GSE155119 data set and biological prediction. Subsequently, the miR-138-5p, CCND1, and circ-CCND1 expression were detected in the non-calcified and calcified aortic valve. Then Pearson correlation analysis was performed to analyze the correlation between the above expression, and dual luciferase and RNA-pull down assays for verifying the target relationship. Porcine aortic valve interstitial cells (AVICs) were isolated and transfected with pcDNA-circ-CCND1, miR-138-5p inhibitor, and miR-138-5p mimics. The alkaline phosphatase (ALP) activity was quantitatively analyzed by ALP staining, and alizarin-red staining was to check the calcium nodules formation. Finally, Western blot was applied to detect the expression of proteins associated with osteogenic differentiation (Runx2, Osterix, OPN) and CCND1/P53/P21 pathway proteins. Circ-CCND1 was highly expressed in calcific aortic valves. After inhibiting circ-CCND1 expression, a significant reduction was shown in ALP activity, the degree of ossification and the formation of calcium nodules in AVICs, and osteogenic differentiation-related protein expression and CCND1/P53/P21 pathway protein expression. By contrast, inhibition of miR-138-5p and circ-CCND1 together promoted the calcification of AVICs and expression of CCND1/P53/P21 pathway proteins. P53 inhibitor (PFT-alpha) could significantly reduce activation of CCND1/P53/P21 pathway protein expression by circ-CCND1 overexpression. However, P53 activator (Nutlin-3) significantly restored the suppression of the above pathway-related protein expression by downregulation of circ-CCND1. Circ-CCND1 sponges miR-138-5p to regulate CCND1 expression, thereby promoting the calcification of AVICs.