The Role And Potential Mechanisms Of LncRNA-MEG3 In Calcific Aortic Valve Stenosis

BackgroundCalcific aortic valve stenosis（CAVS）is a common valve disease in the western world.It is characterized by progressive fibro-calcific remodeling and thickening of the aortic valve leaflets that,over years,evolve to cause severe obstruction to cardiac outflow.China has become a country with the largest elderly population in the world.With the acceleration of population aging,the prevalence of CAVS is increasing year by year.So far,no pharmacotherapy has proved to be effective for patients with CAVS,Hence,surgical or transcatheter aortic valve replacement is the only effective treatment option for severe CAVS.Aortic Valve Interstitial Cells（AVIC）are the predominant cells in the aortic valve.Under pathological stimulation,AVIC undergo a phenotypic transformation from static fibroblasts to osteoblasts,which is regarded as a fundamental step during the CAVS.However,the specific regulatory mechanism of the AVIC osteogenic differentiation has not been clarified.Recently,many studies have shown that long noncoding RNA（lncRNA）can regulate the expression of osteogenic genes at both transcriptional and post-transcriptional levels,thus participating in the pathological process of CAVS.However,the role of lncRNA in the occurrence and development of CAVS is still far from clearObjectiveThis study aimed to screen differentially expressed lncRNAs during CAVS,and then selected the target lncRNA and explore its role in osteogenic differentiation of AVIC.Methods1.Construction and evaluation of CAVS model in mice:Ldlr^-/-mice were fed a western diet for 6 months to induce aortic valve calcific.Control group mice were fed a normal diet.And then both groups were evaluated via cardiac echocardiography,HE staining and von kossa staining.2.Screening of differential expressed lncRNAs and identification of the target lncRNA:RNA-seq was used to screen differentially expressed lncRNAs in aortic valve tissues of CAVS mice and control mice.After verification of sequencing results by RT-qPCR,lncRNA MEG3 was selected as the target lncRNA.3.Establishment and identification of an AVIC calcification model in vitro:Osteogenic medium was used to induce AVIC calcification.Alizarin red staining was used to detect the formation of calcium nodules after mineralization induction for 0d,7d,14d,and 21d.Meanwhile,western blot was used to detect the protein expression level of markers of osteogenic differentiation such as Runx2 and Osterix.4.Investigation the role of lncRNA MEG3 in osteogenic differentiation of AVIC:First,the expression level of lncRNA MEG3after mineralization induction for 0d,7d,14d,and 21d were detected by RT-qPCR.Secondly,Ad-MEG3 and sh-MEG3 recombinant adenovirus were used to infect AVIC individually,so as to overexpress or knock down the expression level of MEG3 in AVIC.RT-qPCR was performed to measure the infection efficiency,and then osteogenic induction for 21days.The protein expression levels of Runx2 and Osterix in each group were detected by western blot,and the formation of calcified nodules was detected by alizarine red staining.5.Investigation the mechanism of lncRNA MEG3 regulating AVIC osteogenic differentiation:Ad-MEG3 was used to infect AVIC to overexpress the expression level of MEG3 in AVIC.AVIC infected with Ad-vector as a control group.RT-qPCR was performed to detect the infection efficiency.AVIC were induced osteogenic differentiation for 21days with or without DKK1,a canonical inhibitor of the Wnt/β-catenin pathway.The protein expression levels of Wnt/β-catenin,Runx2,and Osterix in each group were detected by western blot.Results1.Construction of CAVS model in mice:Echocardiography showed a significant increase in peak aortic valve velocity in CAVS mice,accompanied by an increase in left ventricular wall thickness.HE staining showed aortic valve were significantly thickened in CAVS mice,and von Kossa staining showed a significant increase of calcium deposition in the aortic valve in CAVS mice.2.Screening of differential expressed lncRNAs:RNA-seq results showed that 317 lncRNAs were significantly up-regulated and 176lncRNAs were significantly down-regulated in calcified aortic valves.Lnc RNA MEG3 was selected for subsequent experiments,and RT-qPCR verification results were consistent with sequencing results.3.Establishment and identification of an AVIC calcification model in vitro:The expression levels of Runx2 and Osterix proteins as well as calcified nodules were increased during the mineralization induction and were most significant at day 21.These results indicated that an AVIC calcification model was in vitro successfully established,and 21 days were determined as the optimum induction time for subsequent experiments.4.Investigation the role of lncRNA MEG3 in osteogenic differentiation of AVIC:Lnc RNA MEG3 is up-regulated during AVIC osteogenic differentiation.Overexpression of MEG3 can promote the protein expression of Runx2 and Osterix and the formation of calcium nodules.Conversely,MEG3 knockdown inhibits the protein expression of Runx2 and Osterix,and inhibits the formation of calcium nodules.These results suggest that MEG3 can promote osteogenic differentiation of AVIC.5.Investigation the mechanism of lncRNA MEG3 regulating AVIC osteogenic differentiation:Western blot results showed thatβ-catenin,Runx2 and Osterix expressions were significantly increased in ad-MEG3group compared with ad-vector group.The up-regulation effect caused by MEG3 overexpression can be alleviated by the addition of DKK1.Therefore,lncRNA MEG3 can promote AVIC osteogenic differentiation via the Wnt/β-catenin signaling pathway.ConclusionsLnc RNA MEG3 is significantly upregulated in calcified aortic valve tissues of mice.Lnc RNA MEG3 can promote AVIC osteogenic differentiation via the Wnt/β-catenin signaling pathway.Figures 8,Tables 12,References 50...