The Role Of MiR-22/lncRNA Network In Calcific Aortic Valve Disease And The Underlying Mechanisms

Background Calcific aortic valve disease(CAVD)is a common cardiovascular disease,characterized by a high morbidity and mortality due to ineffective medical prevention and treatment.With the population age,CAVD has become an economic and social burden on aging populations.Therefore,it is very important to explore the cellular and molecular mechanisms in the pathogenesis of CAVD.Once thought purely degenerative in nature,CAVD progression is now believed to be an osteogenesis process that involves the coordinated actions of lipid deposition,inflammation,and phosphate signaling pathway transduction occurring within the aortic valve leaflets.The osteogenic transformation of vascular mesenchymal cells is considered to be the main mechanism of CAVD pathogenesis.The bone formation within the valvular interstitial cells(VICs)is thought to be the main mechanism of CAVD.Non-coding RNA is a class of functional and wide-ranging regulatory elements.Recently numerous studies have shown that multiple mi RNAs are involved in the regulation of bone formation and valvular calcification.However,the role of mi RNAs on calcific valvular disease is still at its infancy.The functions of mi RNAs in the process of osteoblast differentiation remains to be further studied.Long non-coding RNAs(lnc RNAs),whose transcripts are longer than 200 nucleotides,modulate target genes expression at the epigenetic,transcriptional and post-transcriptional levels.Lnc RNAs had been reported to be able to indirectly regulate genes expression by interacting with mi RNAs.Although the role of lnc RNAs in CAVD is still unclear,previous studies have shown that mi RNA interaction with lnc RNAs might play a crucial role in the pathogenesis and prognosis of CAVD.Therefore,the mechanism exploration of mi RNA and mi RNA/lnc RNA regulatory networks in valvular calcification will provide valuable clues to finding therapeutic targets for CAVD.Objectives 1.Screening differentially expressed mi RNAs during aortic valve calcification and identifying target mi RNAs.2.Identifying and elucidating the role and mechanism of mi R-22 in aortic valve calcification.3.Screening the potential mi R-22/lnc RNAs regulatory networks involved in the regulation of CAVD progression.Methods 1.Tissue specimens and clinical baseline a)Procurement and pretreatment of tissues: A total of 45 aortic valve tissue was taken from explanted from CAVD patients(n = 33)and heart transplant procedures as non-CAVD controls(n = 12)between October 2016 to October 2017 at the Department of Cardiovascular Surgery,Changhai Hospital.Half of each tissue sample was used to prepare valve stromal cells or stored in RNAlater(Life Technologies)immediately,and the other half was rapidly fixed in 4% paraformaldehyde or frozen in liquid nitrogen for further tests.b)clinical baseline: The patients' clinical characteristics were collected,including medical history,laboratory values and echocardiographic data.2.Isolation and culture of human aortic valvular interstitial cells(HAVICs)a)Isolation of human aortic valvular interstitial cells(HAVICs): HAVICs were isolated from normal and calcified valves and was digested by modified collagenase methods.Cells were resuspended and cultured in DMEM medium,supplemented with 100 U/m L penicillin,100 ?g/m L streptomycin,and 10% fetal bovine serum,in an incubator with 5% CO2 at 37 °C.Cells of passages 3 to 6 were used for the experiments.b)Confirmation of HAVICs: The purity of isolated HAVICs was obtained by immunofluorescence assay through detecting the expression interstitial cell marker Vimentin and endothelial cell marker CD31.c)In vitro calcification models: HAVICs were cultured with DMEM complete medium supplemented with 10-7 mol/L insulin,50 ?g/m L ascorbic acid,2 mmol/L Na H2PO4 and 5% fetal bovine serum for up to 7 days to establish calcification model in vitro.3.Detection of calcium deposits and osteogenic differentiation related markersa)Detection of calcium deposits: The Alizarin red staining,Von Kossa staining,Calcium Assay,and alkaline phosphatase activity assay were used to detect the calcification levels of valve tissues.b)Detection of osteogenic differentiation markers: q PCR and western blot assay were applied to detect the expression of osteogenic markers(OPN,and Runx2),respectively.4.The role of mi R-22 in aortic valve calcificationa)mi R-22 expression level: Real-time PCR was applied to detect the expression level of mi R-22 in calcified valves tissues and in VICs after calcification induction.Fluorescence in situ hybridization assay was used to confirm the region of mi R-22 in calcified valves.b)mi R-22 function study: In order to overexpress and knockdown mi R-22 expression,Ad-mi R-22,Ad-Scramble and shmi R-22,sh Scramble were transfected into HAVICs,respectively.After identifying the transfection efficiency,HAVICs were treated with calcification induction to investigate the role of mi R-22 in aortic valve calcification.5.The mechanism study of mi R-22 during osteogenic differentiation of VICsa)Identify new targeted gene: Bioinformatics analysis was used to predict and screen the targeted genes of mi R-22.Dual-luciferase reporter assay and western blot assay were applied to verify the relationship between mi R-22 and targeted genes.Then,the rescue experiment was carried out to verify the authenticity of the targeted genes.b)Targeted gene function study: Immunohistochemistry,real-time PCR and western blot assay were used to detect the expression of the targeted genes in VICs after calcification induction.Adenovirus mediated gain-and loss-of-function study was applied to investigate the function of targeted genes on VICs osteogenic differentiation.6.Screening the potential mi R-22/lnc RNAs regulatory networksTotal RNA was extracted from the normal and calcified valvular tissues.The differentially expressed lnc RNAs were screened by a lnc RNA microarray.Then bioinformatics analysis was applied to screen differentially expressed lnc RNAs contained potential binding sites of mi R-22 and to further explore the mi R-22/lnc RNA regulatory networks in regulation of valvular calcification.Results 1.The differentially expressed mi RNAs during aortic valve calcificationTo identify the aberrant expression of mi RNAs in calcified aortic valve,real-time PCR was applied to detect the expression profiles of osteogenic mi RNAs in a small cohort of CAVD patients.Compared with control group,mi R-21,mi R-22,and mi R-125a/b were significantly increased,while mi R-10,mi R-214,and mi R-204 were significantly decreased in CAVD group.Furthermore,the highly expressed mi R-22 in calcified aortic valves was confirmed in a larger cohort of CAVD patients.Fluorescence in situ hybridization assay confirmed that mi R-22 was observed throughout the regions of the calcified valves,and was localized in VICs,as indicated by the co-expression of vimentin.2.Functional study of mi R-22 during osteogenic differentiation of VICsTo clarify the function of mi R-22 on VICs osteogenic differentiation,adenovirus mediated gain-and loss-of-function studies were applied.As analyzed by Alizarin Red S staining,the significant increased calcium nodules could be observed in mi R-22-ovexpressing VICs after calcification induction.Meanwhile,the overexpression of mi R-22 resulted in further increases in calcium deposition,ALP activity,m RNA and protein levels of osteoblastic differentiation markers in VICs.In contrast,silencing of mi R-22 in VICs negated the osteogenic medium-induced increase in calcified nodule formation,calcium deposition,ALP activity,expression of osteoblastic differentiation markers.3.Mechanism of mi R-22 in regulating osteogenic differentiation of VICsBy bioinformatics analysis,dual-luciferase reporter assay and rescue experiments,CAB39 was demonstrated to be putative target of mi R-22.Immunohistochemistry,real-time PCR and western blot assay found that the expression of CAB39 was significantly decreased in VICs after calcification induction.Correlation analysis showed that CAB39 expression was negatively correlated with the severity of valve calcification.Then,adenovirus mediated gainand loss-of-function study was applied to investigate the function of CAB39 on VICs osteogenic differentiation.Overexpression of CAB39 could significantly decrease calcium nodules and deposition,which suggested that CAB39 might play a protective role in CAVD progression.Furthermore,we investigated the role of mi R-22 on CAB39/STRAD/LKB1 complex and its downstream AMPK/m TOR signaling pathway.The results showed that mi R-22 promoted the osteogenic differentiation of VICs by regulating CAB39-LKB1-AMPK-m TOR signaling pathway.4.The potential mi R-22/lnc RNAs regulatory networks during osteogenic differentiation of VICsA lnc RNAs microarray was applied to detect the differential expressed lnc RNAs during osteogenic differentiation of VICs.A total of 253 lnc RNAs were identified significantly differentially expressed,including 70 upregulated lnc RNAs and 183 downregulated lnc RNAs.Bioinformatics analysis further showed that 36 lnc RNAs contained potential binding sites of mi R-22,with 5 significantly differentially expressed lnc RNAs from microarray data.Among them,expression of u lnc-STX6-2,NEAT1,and SNORA67 were significantly increased,whereas expression of lnc-MLLT1-2,and lnc-C5orf42-2 were significantly decreased.The exact roles of these differentially expressed lnc RNAs remained to be investigated in future.Conclusions The expression of mi R-22 was increased during aortic valve calcification and mi R-22 might be a potential inducer of CAVD by inhibiting the CAB39/ AMPK/ m TOR signaling pathway.The results suggested that mi R-22 might serve as a potential therapeutic target for CAVD.