LncRNA MEG3 promotes aortic valve calcification by inducing osteoblast differentiation via Wnt catenin signaling pathway

Abstract Background Calcific aortic valve stenosis (CAVS) is the most common valvular heart disease in elderly population. This study aimed to investigate the role of long non-coding RNAs (lncRNAs) in the development of CAVS. Methods ldlr-/-mice were feeding with western diet for 6 months to induce CAVS. RNA-seq analysis was performed to identify differentially expressed molecular signaling between aortic valves of CAVS mice and control mice. The degree of aortic stenosis was detected by ultrasound. Calcium salt deposits were detected by Haematoxylin and eosin (H&E) staining, Von Kossa staining and alizarin red S staining. PCR and western blotting were employed to detect osteogenic differentiation markers after various interventions. Results A mouse CAVS model was successfully established, as evidenced by increased thickness and calcium deposition in the aortic valve leaflets and increased transvalvular peak jet velocity. RNA-seq analysis showed that lncRNA MEG3 was significantly enriched in aortic valves of CAVS mice as compared to that of control mice. MEG3 expression in aortic valve, assessed by RNA in situ hybridization and PCR, was also significantly upregulated during the osteoblast differentiation of AVICs in vitro. Gain- and loss-of-function experiments indicate that MEG3 could indeed promote osteoblast differentiation of primary AVICs as evidenced by increased calcified nodule formation and expressions of osteoblast differentiation markers (Runx2 and osterix). Consistent with these in vitro data, MEG3 knockdown in vivo through tail injection of antisense oligonucleotide chain MEG3 (ASO-MEG3) at 6 weeks interval for 12 weeks significantly attenuated aortic valve calcification in CAVS mice. Subsequent KEGG analysis demonstrated that the Wnt/β-catenin signaling pathway was significantly enriched during the CAVS progression. Overexpression of MEG3 activated the Wnt/β-catenin signaling pathway and upregulated osteogenic protein expressions, while this upregulation could be significantly ameliorated by Dickkopf 1, a Wnt/β-catenin pathway inhibitor. Conclusion Present study demonstrates that LncRNA MEG3 could promote aortic valve calcification by inducing osteoblast differentiation via activating Wnt/β-catenin signaling pathway. Our results provide experimental evidence of targeting MEG3 and Wnt/β-catenin signaling pathway as potential promising therapeutic option for prevention and treatment of aortic valve calcification.