The Effects And Mechanism Of MicroRNA-138 On Osteoblastic Differentiation Of Valvular Interstitial Cells In Degenerative Calcific Aortic Valve Disease

Calcific aortic valve disease(CAVD),including early valve thickening and advanced valve stenosis,closed increasing progressive disease,can cause malignant arrhythmia,heart failure or even sudden death,seriously threaten human health.With the improvement of living standards and population aging,calcific aortic valve disease(CAVD)has become an serious clinical issues faced by cardiologists.The early symptoms of CAVD include valve thickening and sclerosis,and late manifestations include valve dysfunction and narrow.It can cause arrhythmia,heart failure,thromboembolism and even sudden death,which seriously threatens human physical and mental health.With the increasing number of the aging population in our country,DCAVD cause great threaten to elderly.Until now,there is no effective treatment for CAVD,and aortic valve replacement is still recognized as the most effective treatment.However,the existing artificial valves all have their own defects,and mechanical valves require lifelong oral warfarin anticoagulation,which is prone to bleeding or embolism and other adverse events.Biological valve is easy to decline,short life,especially the elderly patient's condition is generally relatively complex,relatively more complications,high surgical risk.Therefore,exploration of the pathogenesis of CAVD and searching the potential treatment targets to control the process of valve calcification have important clinical significance.Valvular interstitial cells are the main cells regulating the structure and function of the aortic valve.The transdifferentiation of VICs to osteogenic phenotype may be related with the formation of calcific nodules.The increasing expression of osteogenic factors such as alkaline phosphatase(ALP),OPN,BMP2,MSX,Runx2 have been detected in CAVD valve samples.These osteogenic factors accumulating around the apoptotic or necrotic VICs induce the calcium nodes formation,eventually leading to an irreversible calcification process.The above study indicated that VICs differentiating into osteoblasts may provide an important basis for the development of DCAVD,and exploring the pathogenesis of osteogenic differentiation of VICs can provide a new strategy for preventing heart valve calcification.MicroRNAs are important short non-coding regulatory molecules in cells,which can participate in regulating many physiological and pathological processes.It can sequence-specifically bound to the mRNA 3'-UTR(Untranslated region,UTR)region,and finally causing mRNA degradation or translation inhibition.MicroRNA 138(miR-138)was reported to be invoved in several biological processes,including developmental events tied to cell differentiation.A study revealed that miR-138 demonstrated protective effects on hypoxic cardiomyocytes.Besides,miR-138 was confirmed to have an important regulatory role in osteoblast differentiation and bone formation.Recent studies have found that miR-138 is a kind of mechanically sensitive miRNAs which can influence osteoblast differentiation,while miR-138 blocking can effectively reverse the inhibitory effect of mechanical unloading on osteoblast differentiation.In the view of the dual regulatory effects of miR-138 on osteogenic differentiation and cardiovascular function,the expression and function of miR-138 in cardiac valve disease are still under investigation.In the present study,we explored the role of miR-138 in the aortic valve calcification in CAVD,and tested the hypothesis that upregulation of miR-138 would inhibit aortic valve calcification by cell studies and animal models,which informs a novel direction for treating CAVD.Section 1 miR-138 expression in DCVAD tissuesObjective:To investigate the expression of miR-138 in DCVAD tissues.Materials and Methods:The calcified degenerative aortic valve resected during valve replacement surgery was collected as the experimental group.In order to reduce the error caused by individual differences,the relatively smooth non-calcified part of the patient's surgically removed valve was used as the control group.HE staining was used to evaluate the valve structure.qRT-PCR was performed to analysis miR-138 expression in the valve tissues of the experimental group and the control group.Results:HE staining indicates the proliferation of collagen fibers in the valve tissue of degenerative calcification disease,and there is obvious calcium deposition.The qRT-PCR results showed that the expression of miR-138 in the calcified valve tissue of DCAVD patients was significantly lower than that of the surrounding relatively normal smooth valve tissue.Summary:MiR-138 was down-regulated in DCVAD tissues,and involved in the pathogenesis of CAVD.Section 2 miR-138 inhibits VIC osteogenic differentiationObjective:To investigate the regulation effects of miR-138 in VIC osteogenic differentiationMethods:(1)VIC isolation and identification:Normal aortic valves were obtained from the acute Stanford A aortic dissection patients.The endothelial layer of the aortic and ventricular aspects were removed,and the non-leaflet tissues were carefully eliminated.Then the collected valves were immersed in 0.25%trypsin and cut into pieces for digestion.After that,primary VICs were obtained and seeded in MEM medium for cell culture.(2)Establishment of osteogenic induction model in vitro.After transfection,the cultured cells were induced osteogenic differentiation in OM.(3)Calcification and osteogenic differentiation analysis.Alizarin red calcium staining calcium deposition determination and alkaline phosphatase activity detection.Osteogenic differentiation detection:QRT-PCR was used to detect the mRNA expression changes of osteogenic markers RUNX2,MSX2 and ALP.(4)The functional verification of miR-138 in the calcification model of interstitial cells of the valve by qRT-PCR.Transfect miR-138 inhibitors,inhibitor NC,miR-138 mimics and mimics NC into VICs respectively to obtain VICs that overexpress or knock down miR-138,measure the transfection efficiency and then induce calcification.Verify miR-138 by the above-mentioned experimental detection method.Transdifferentiation effect on VICs osteogenic.Results:(1)Cell phenotype identification was performed in VIC.Immunohistochemical results revealed the positive staining for ?-SMA and vimentin in VICs(60%-70%and 100%,respectively).(2)After 3 days of calcification induction,decreased expression of miR-138 were detected in the VICs.(3)Up-regulated intracellular miR-138 levels were observed followed by an miR-138 mimic transfection,and down-regulated miR-138 expression were recorded in miR-138 inhibitor group.(4)The expressions of Runx2,MSX2,and ALP were detected after transfection with an miR-138 mimic and inhibitor.The results showed that the miR-138 mimic significantly suppressed mRNA expression of Runx2,MSX2,and ALP while miR-138 inhibitor promoted the expression of Runx2,MSX2,and ALP after 7 days of calcification induction.ALP activity was also detected and miR-138 overexpression significantly repressed ALP activity,while miR-138 inhibitor increased ALP activity after 14 days of osteogenic differentiation 14 days.In addition,alizarin red staining was used to analyze calcium nodule formation.At the 14th day of calcification induction,decreased calcium nodules were observed in the miR-138 overexpression group,while increased calcium nodules were observed in the miR-138 low-expression group.These data demonstrated that miR-138 plays a role in inhibiting VIC osteogenic differentiation.Summary:MiR-138 inhibits VIC osteogenic differentiation.Section 3 Mechanism analysis of miR-138Objective:To clarify the mechanism of miR-138 in the osteogenic differentiation of VICs.Methods:(1)Screening of target genesThe potential targets of miR-138 were speculated using bioinformatics methods(miRanda and Targetscan).Based on their intersection,genes that could be negatively regulated by miR-138 were screened.Further,the genes related to osteogenic differentiation and aortic valve function were screened,and their potential targets were predicted.(2)Verification of target genes? Double-luciferase reporter gene assay were used for the evaluation of the relationships between miR-138 and target genes.? VICs were transfected with miR-138 inhibitors,inhibitor NC,miR-138 mimics and mimics NC,respectively,and the effect of miR-138 expression on target gene levels was analyzed.? The authenticity of target genes was verified by a complement experiment.(1)Functional analysis of target genesAdenoviruses with target gene overexpression and knockdown were constructed.After transfection,the osteogenic differentiation of VICs was induced,and the effects of target genes on the osteogenic differentiation of VICs were detected.(2)In vivo study of miR-138 on aortic valve calcification? Establishment of animal model.An animal model of calcification was established according to the modified Price method.The specific dosage and usage of the drugs are as follows.The mice in the two experimental groups were injected with agomir-138 and agomir-138 NC respectively,with the dose of 5 mg/kg.Vitamin D3 was injected subcutaneously to the mice in the three groups except the blank control group with the dose of 500,000 IU/kg to induce soft-tissue calcification,once a day for 3 consecutive days.? evaluation of blood flow velocity and aortic valve pressure difference.The maximum transvalvular blood flow velocity and the maximum transvalvular pressure difference of the animals were measured using an ultrasound Doppler flow meter.? Relevant detection of osteogenic differentiation.The changes in the mRNA expressions of osteogenic markers RUNX2,MSX2 and ALP were detected by qRT-PCR,respectively.? Expression analysis of miR-138 and its target genes.The expression levels of miR-138 and target gene FOXC1 in each group were measured by qRT-PCR,respectively.Results:(1)To clarify the molecular mechanism,among the candidate sequences,we found a highly conserved and specific combination sequence between miR-138 and FOXC1 3'UTR.(2)miR-138 mimic significantly repressed luciferase activity when cotransfected with reporter containing WT FOXC1 3'UTR but not MT FOXC1 3'UTR.(3)The synthetic mimic and inhibitor of miR-138 were transfected into VICs.The results showed that miR-138 overexpression significantly inhibited FOXC1 mRNA expression,while the miR-138 inhibitor promoted the expression of FOXC1.As shown by Western blotting,FOXC1 protein expression was significantly decreased by the miR-138 mimic at day 7 after calcification induction.The results of Western blotting were consistent with the quantitative PCR results.The above results indicated that miR-138 directly targets FOXC1.(4)To confirm that the effect of miR-138 during VIC osteogenic differentiation is mediated by targeting FOXC1,we transfected an miR-138 mimic into VICs after FOXC1 overexpression,and then proceeded with osteogenic differentiation.The results showed that miR-138 mimic-FOXC1 overexpression significantly inhibited mRNA expression of Runx2,MSX2,and ALP at day 7 after calcification induction and ALP activity at day 14 after calcification induction.Decreased calcium nodules were observed in the miR-138 mimic-FOXC1 overexpression group at day 14 after calcification induc-tion.The results demonstrated that miR-138 regulates VICosteogenic differentiation through FOXC1.(5)FOXC1 overexpression promoted osteogenic differentiation of VICs,indicated by mRNA expression of Runx2,MSX2,and ALP at day 7 after calcification induction,and ALP activity and alizarinred staining at day 14 after calcification induction,while Si-FOXC1 suppressed osteogenic differentiation of VICs,as indicated by mRNA expression of Runx2,MSX2,and ALP,ALP activity and alizarin red staining.(6)Decreased miR-138 expression were detected in vitamin D 3-treated models.(7)Compared with control group,intra aortic ring velocity and transvalvular pressure gradient were significantly increased in mice treated with vitamin D3.However,mir-138 overexpression significantly reduced the velocity and transvalve pressure gradient in the aortic ring.(8)Agomir-138 significantly suppressed the mRNA expression of Runx2,MSX2,and ALP in the vitamin D3-treated mice.(9)Agomir-138 significantly down-regulated the mRNA expression of FOXC1.Summary:MiR-138 would inhibit aortic valve calcification by reducing FOXC1.ConclusionsThe development of DCAVD can be accompanied by the decreased expression of miR-138 in valve tissue,and similar phenomena were also observed in the VICs model in vitro.Functional experiments revealed that miR-138 could block the process of osteogenic differentiation in VICs.Using bioinformatics analysis,luciferase reporter and In vitro rescue experiment assays,FOXC1 was confirmed to be the target gene of miR-138,which can be negatively regulated by miR-138.Our studies revealed that miR-138 could block the process of osteogenic transdifferentiation by targeting FOXC1,thereby inhibiting the development of CAVD.