The Role And Mechanism Of MiR-214 In Calcific Aortic Valve Disease

BackgroundCalcific aortic valve disease?CAVD?is a common heart valve disease in cardiovascular surgery,with valvular fibrosis and calcification as its pathological characteristics.At present,there is no pharmacotherapy scheme available for these patients.Aortic valve replacement is the only choice when these patients are present with obvious clinical symptoms and hemodynamic disorder.Expensive fee and high perioperative risk are two inevitable difficulties that these patients often encounter.Therefore,research on the pathogenesis of CAVD and treatment therapy has been to be the focus of CAVD research.Current researches have confirmed that CAVD is a pathological process involving multiple factors and can be divided into two phases,including the initiation phase and the progress phase.The aortic leaflets can gradually progress to aortic valve sclerosis during the initiation phase,and its pathological process is similar to that of atherosclerosis,including endothelial damage,lipid deposition,and subsequent inflammatory reactions.The advanced stage is mainly with the manifestation of extracellular fibrosis and calcification.The differentiation of valve interstitial cells?VICs?to osteoblasts may act as the theoretical basis of valvular ectopic osteogenesis.Various osteogenic related proteins such as runt-related transcription factor 2?Runx2?,Sp7,Activating Transcription Factor 4?ATF4?,osteopontin?OPN?,and osteocalcin?OCN?have been demonstrated to be expressed positively in CAVD valve samples.Micro RNAs are a series of non-coding RNA with a length of about 18-26 bp.It can silence the expression of its target gene through post-transcriptional regulatory mechanisms,which consist of the way by hampering the translation process of messenger RNA?m RNA?or promoting the degradation of m RNA.Recently,mi R-214has been proved to regulate the physiological differentiation and formation of bone tissue,as well as pathologic change of bone.Its expression pattern is quite conserved in vertebrates,and it maintains the homeostasis of bone tissue by regulating the activity of osteoblasts and osteoclasts.In addition,mi R-214 can be used as a targeted drug for the treatment of bone diseases and has been proven successful in animal experiments.The regulatory function of micro RNA in CAVD is still in the preliminary exploration stage.In view of the ectopic osteogenesis process of aortic valve in CAVD,the imbalance of mi R-214 expression may be related to the initiation and development of CAVD.Recent studies have confirmed that mi R-214 is significantly downregulated in calcified aortic valves,while the expression of osteogenic related proteins has been significantly upregulated.However,the function and mechanism of mi R-214 in CAVD remain unclear.Therefore,exploring the function and mechanism of mi R-214 in CAVD is vital for revelation of the pathogenesis of CAVD,and may provide the theoretical basis for mi R-214 as a targeted drug for CAVD.Objectives1.To identify the differential expression level of mi R-214 and osteogenic related proteins in the CAVD process.2.To clarify the function and mechanism of mi R-214 in the osteogenic differentiation of VICs.3.To identify the function of mi R-214 in CAVD animal models in vivo.MethodsA?Histological and molecular biology analysis of human aortic valve1.Collection of clinical specimens:Aortic valve specimens from CAVD patients underwent aortic valve replacement were collected,specimens from heart transplantation recipients were collected as negative control.All specimens were stored in 10%formalin solution and liquid nitrogen separately for further research.2.Detection of osteogenic related proteins:Alizarin red staining and HE staining were used to confirmed the pathological change of aortic valve.The expression level of osteogenic related proteins in both groups were analyzed by Western Blotting and immunohistochemistry.3.Detection of mi R-214:The expression level of mi R-214 in two groups was detected by real-time quantitative PCR.B?Isolation and identification of VICs,establishment of osteogenic induction model in vitro1.Isolation of VICs:VICs were isolated from the aortic valve of heart transplant recipients by type II collagenase digestion process.VICs in 2^ed-5^th generation after passage were used for cytological study.2.Identification of VICs:The expression of Vimentin,?-SMA,and CD31 were tested by immunofluorescence.3.Establishment and identification of in vitro osteogenic induction model of VICs:VICs were cultured and stimulated by osteogenic induction medium,which consist of 2m M Na H₂PO₄,50?g/m L ascorbic acid,and 10^-7 mol/L insulin.Real-time quantitative PCR and Western Blotting were used to detect the expression level of osteogenic related proteins in VICs by time gradient.Alizarin Red staining was used to confirm the calcium deposition.4.Detection of mi R-214 in osteogenic induction model in vitro:The expression level of mi R-214 in VICs at different time point was detected by real-time quantitative PCR.C?The function and mechanism analysis of mi R-214 on osteogenic differentiation of VICs1.Function study of mi R-214:The agomir-214 and antagomir-214 were transfected into VICs,and then the transfection efficiency was assessed by real-time quantitative PCR.The function of mi R-214 on osteogenic differentiation of VICs was assessed by real-time quantitative PCR,Western Blotting,and Alizarin Red staining.2.Mechanism analysis of mi R-214:bioinformatics analysis was used to predict the putative binding site of mi R-214,dual-luciferase reporter assay and rescue experiment were used to verify the regulatory mechanism.D?The function analysis of mi R-214 knockout in CAVD rat model1.Identification of rat genotype:agarose electrophoresis and real-time quantitative PCR were used corporately to identify neonatal rat genotype.2.Establishment and identification of CAVD rat model:the combination of Western diet with intraperitoneal injection of vitamin D?25000IU/kg?for 4 months was used to construct CAVD model.HE staining and alizarin red staining were used to identify the modeling effect.3.Function analysis of mi R-214 knockout in vivo:rats were devided into 3 groups,including wild-type,heterozygote,and mutant type group.Echocardiography was used to evaluate the degree of aortic stenosis.Alizarin red staining was used to assess the calcium deposition.The expression level of osteogenic related proteins in aortic valve was determined by immunohistochemistry.ResultsA?Histology and molecular biology analysis of human aortic valveThe expression level of Runx2,Sp7,OPN and ATF4,which are crucial osteogenic transcription factors,were significantly upregulated in CAVD valve samples contrast to that of negative control by Western Blotting and immunohistochemistry analysis.In addition,the real-time quantitative PCR result showed that mi R-214 was significantly downregulated in calcified aortic tissues.B?Isolation and identification of VICs,establishment of osteogenic induction model in vitroHuman aortic VICs were successfully obtained by 2 steps collagenase digestion process.The purity of VICs was confirmed by immunofluorescence with positive staining of Vimentin and?-SMA.No mixture of endothelial cell was observed since the immunofluorescence staining of CD31 was negative.The expression level of osteogenic related proteins,including Runx2,Sp7,ATF4,and OPN,was significantly upregulated from day 3 after treatment with osteogenic induction medium,which was detected by real-time quantitative PCR and Western Blotting.Alizarin red staining confirmed that calcium deposition could be detected at day 7 after osteogenic induction.Furthermore,the real-time quantitative PCR revealed that the expression level of mi R-214 was significantly downregulated from day 1 after osteogenic induction in VICs.C?The function and mechanism analysis of mi R-214 in osteogenic differentiation of VICsThe overexpression and knockdown of mi R-214 in VICs were confirmed by real-time quantitative PCR after transfected with agomir-214 and antagomir-214,respectively.As shown in PCR and Western Blotting analysis,the expression level of osteogenic related proteins was inhibited by mi R-214-overexpressing.In contrast,the expression level of osteogenic related proteins was significantly upregulated after silencing of mi R-214.Alizarin red staining confirmed that overexpression of mi R-214alleviated calcium deposition of VICs after osteogenic induction,while silencing of mi R-214 accelerated calcium deposition of VICs.As shown in bioinformatics analysis,the 3'-UTR of ATF4 and Sp7 were putative binding site of mi R-214.The 3'-UTR of ATF4 and Sp7 were verified to be targeted binding site of mi R-214 by dual-luciferase reporter assay.Furthermore,the downregulation of ATF4 or Sp7 by si RNA alleviated the calcium deposition promoted by antagomir-214 in VICs as shown in rescue experiment.D?The function analysis of mi R-214 knockout in CAVD rat modelThe neonatal rat genotype was identified by agarose electrophoresis and real-time quantitative PCR analysis corporately.The positive staining of alizarin red staining at aortic valve suggested that the combination of Western diet with intraperitoneal injection of vitamin D for 4 months could successfully construct rat CAVD model.As shown in echocardiographic analysis,the aortic valve leaflets of mi R-214 knockout rats were thicker compare to that of wide type and heterozygotic rats,accompanying with higher transvalvular pressure gradient and velocity.Aortic valve calcification in mi R-214knockout rats was revealed by Alizarin red staining.The expression level of Runx2,Sp7,OPN,and ATF4 was significantly upregulated in mi R-214 knockout group contrast to that of wide type and heterozygotic group,which was demonstrated by immunohistochemistry,suggesting that mi R-214 knockout promoted aortic valve calcification.ConclusionMiR-214 imbalance in aortic leaflets is responsible for the progress of CAVD.Downregulation of mi R-214 eliminates its inhibitory effect on translation of Sp7 and ATF4,which both are proved to be pivotal osteogenic transcription factor,thereby promoting the development of CAVD.These results suggested that mi R-214 might provide an avenue for treating CAVD.