| With the aging of the population,the incidence of calcific aortic valve disease(CAVD)is increasing year by year,and it has become the most common heart valve disease.In addition,this imposes a huge burden on clinical treatment and socioeconomics.At present,many large-scale clinical trials on the treatment and prevention of CAVD with statins and ACEi drugs have not achieved positive results.Therefore,in the absence of effective drug interventions,in-depth research on the pathogenesis of the disease,the search for new drug effects,and the search for new drug targets have extremely important scientific significance and clinical translational value for the treatment of calcific active diseases.Early studies believed that CAVD was a passive process related to valve degeneration and calcium deposition.Recent studies have found that the occurrence of CAVD is actually a process involving endothelial damage,cell differentiation,lipid infiltration,chronic inflammation,matrix remodeling,and progressive disease.The active process of complex changes such as calcification and neovascularization play an important role in this series of pathophysiological processes in the development of the disease.In particular,differentiation of valve interstitial cells into an osteoblast-like phenotype is considered to be an essential step in valve calcification.In recent years,with the deepening of research,some important molecules and signaling pathways that regulate the transformation of valve interstitial cells into osteoblast-like cells have gradually been discovered,such as Notch1,MAPK signaling pathway,BMP2 signaling pathway,RANK/RANKL/OPG pathway,Wnt/β-catenin pathway,etc.These molecules and signaling pathways play an important role in the occurrence and development of CAVD.In particular,the BMP2 signaling pathway is the most classic osteogenic differentiation pathway.It is an important member of the TGF-βsuperfamily.Its main role is to stimulate the formation of osteoblasts and promote the osteogenic differentiation of various cells.In valve interstitial cells,BMP2 activates downstream pSmad1/5/8 by binding to its receptors,and then upregulates various protein factors of osteogenic differentiation,resulting in valve calcification.Long noncoding RNAs(lncRNAs)are a class of transcripts longer than 200 nucleotides and generally do not have the ability to encode proteins.Previous studies have called lncRNAs "noise" in the genome.In recent years,with the deepening of research,it has been found that lncRNAs play important functions in the development of various organs,physiological activities,and the development of diseases.In general,lncRNAs can play a regulatory role in various processes of gene expression from transcription initiation to protein translation activation.In the process of aortic valve calcification,multiple lncRNAs have been found to play important functions,such as Hotair,TUG1,H19,MALAT1,etc.These studies have complemented the understanding of the pathogenesis of CAVD from long noncoding RNAs.With the development of transcriptomics that can provide more accurate digital signals,higher detection throughput and wider detection range,and the continuous improvement of reference genomes,transcriptomics technology based on high-throughput sequencing platforms has allowed us to discover More transcripts of lncRNAs that play important functions in disease.However,compared with coding genes,we still know little about the pathogenesis and role of lncRNAs in CAVD.Especially in recent years,RNA intervention methods have become more and more abundant,and RNA vaccines and oligonucleotide drugs have been verified by clinical trials.RNA as a target for intervention is of great scientific significance and translational value for exploration.The purpose of our study was to use human aortic valve disease samples and normal high-throughput transcriptomic techniques to discover novel long non-coding RNAs that play an important role in CAVD.Constructing animal models of CAVD through high-fat diet feeding,verifying the function of the screened lncRNAs in animal models,and verifying whether the molecules can be used as targets for CAVD intervention through transgenic or oligonucleotide drugs.By extracting primary aortic valve interstitial cells,building a calcification model at the cellular level,and exploring the role and mechanism of target lncRNAs in CAVD through molecular genetic techniques,it provides an important theoretical basis and experimental strategy for disease placement.The expression and regulation of lncRNA SNHG3 in CAVD are summarized as follows:Part Ⅰ:Expression of Long Non-coding RNA SNHG3 in Calcific Aortic Valve DiseaseBackground:Calcific aortic valve disease(CAVD)is a disease that seriously threatens cardiovascular health worldwide,and its pathogenesis is unknown.Long noncoding RNA(lncRNA)is a class of nucleotide RNA transcripts longer than 200 bp.LncRNA may be a valuable therapeutic target for the prevention and treatment of CAVD disease.This study intends to explore the differential lncRNAs of lncRNA in calcific aortic valve disease transcriptome sequencing.Methods:The clinical samples of 10 calcified aortic valves and 12 normal valves were collected for transcriptomic sequencing;differential genes were analyzed by bioinformatics,and human samples were expanded for polymerase chain reaction(PCR)verification.Primary valve interstitial cells(VICs)were extracted from non-calcified aortic valves of transplant recipient hearts and identified by immunofluorescence costaining of α-SMA and Vimentin,cultured for calcification,and knocked out in candidate lncRNAs.Reduced functional screening target lncRNA,SNHG3.ApoE-/-mice were fed a high cholesterol-cholesterol diet for 12 weeks,16 weeks,20 weeks,and 24 weeks to establish a CAVD model.The degree of aortic valve stenosis was detected by ultrasound,calcium deposition was detected by H&E staining,von Kossa staining,Alizarin red S staining,and osteogenic differentiation markers were detected by PCR to describe the degree of aortic valve calcification.The expression of SNHG3 in aortic valve was detected by RNA in situ hybridization and PCR.Results:Transcriptomic analysis found that 33 lncRNAs were significantly upregulated and 39 were significantly down-regulated in calcified aortic valve.Significantly up regulated the top 10 lncRNAs with different folds,and 7 lncRNAs were verified after expanding the sample.Among them,after SNHG3 was knocked down in promoting calcification cultured VICs,the transcription levels of calcification markers ALP,RUNX2,and OPN were most significantly reduced.Therefore,SNHG3 lncRNA as the target of our study.In situ hybridization of VICs showed that SNHG3 was mainly distributed in the nucleus.After calcification-promoting culture of VICs,it was found that the transcription level of SNHG3 was up-regulated with calcification-promoting time and positively correlated with osteogenic differentiation markers.By constructing a calcified aortic valve model in mice,the expression level of SNHG3 was detected,and it was found that the expression of SNHG3 increased significantly with the detection of osteogenic differentiation markers such as ultrasound,pathology and PCR,indicating that the degree of aortic valve calcification was more severe.Conclusions:In this study,transcriptional sequencing of human aortic valve samples was used to screen out the lncRNA SNHG3 related to aortic valve calcification,and to construct a CAVD model to verify that SNHG3 was significantly elevated in CAVD at the pathological and molecular levels and was associated with the osteogenic differentiation of VICs.Part Ⅱ:Long noncoding RNA SNHG3 promoted Osteoblast differentiation of Valvular Interstitial Cells and Calcific Aortic Valve DiseaseBackground:Long non-coding RNA(lncRNA)SNHG3 is highly expressed in humans,animal models,and osteogenic differentiated valvular interstitial cells(VICs);The causal relationship between calcific aortic valve disease(CAVD)still lacks critical direct evidence,and its role in the development of CAVD is still unknown.It is necessary to demonstrate the role of SNHG3 in calcification in VICs and animal models by means of SNHG3 gain or loss of function.role in CAVD.Methods:Primary cells were extracted from the non-calcified aortic valve of transplant recipients,induced by calcification-promoting medium,then knocked down with antisense oligonucletide(ASO)and overexpressed SNHG3 with adenovirus and detected with VICs Osteogenic differentiation-related indicators,alkaline phosphatase(ALP)activity,cell supernatant calcium ion concentration,Alizarin Red S staining to detect the formation of calcium nodules in the cell matrix,and Western blotting to detect cell osteogenic differentiation markers The changes in the levels of Osteopontin,Runx2 and Osteocalcin were used to evaluate the role of this lncRNA in the osteogenic differentiation of VICs.High cholesterol lipid-fed ApoE-/-mice were used to construct a CAVD model,and the antisense oligonucleotide chain knockdown SNHG3(ASOSNHG3)was injected from the tail vein at 12 weeks for intervention,and the control group was injected with ASO-NC for 12 weeks,Ultrasound detection of peak transvalvular aortic flow velocity and aortic valve area,pathological detection of hematoxylin and eosin staining to observe valve morphology,Alizarin Red S staining and von Kossa staining to evaluate valve calcium deposition and formation of calcium nodules;RNA in situ The transcription level of SNHG3 and the expression of calcification markers(ALP,OPN,RUNX2)were evaluated by hybridization and polymerase chain reaction(PCR).These methods were used to elucidate the role of SNHG3 in the development of CAVD and whether it could serve as a molecular target for intervention.RESULTS:Compared with the control group,knockdown of SNHG3 could significantly reduce osteogenic differentiation-related indicators,ALP activity,calcium ion concentration in cell supernatant,calcium nodule formation,and osteogenic differentiation-related proteins after VICs calcification-promoting culture.However,overexpression of SNHG3 can significantly increase osteogenic differentiation-related indicators.After SNHG3 knockdown intervention in the mouse CAVD model,compared with the control group,the peak trans-aortic valve flow velocity was reduced,the aortic valve orifice area was restored,the valve leaflet thickness was reduced,the calcium salt deposition and calcium nodules were reduced,and calcification markers expression decreased.Conclusion:SNHG3 can promote the osteogenic differentiation of VICs,thereby promoting the progression of CAVD.Knockdown of SNHG3 intervention in a CAVD model can significantly reduce the progression of CAVD,and SNHG3 may serve as a new target for the disease.Part Ⅲ:The Melocular Mechanism of Long Noncoding RNA SNHG3 Promoting Calcific aortic valve diseaseBackground:Long non-coding RNA(lncRNA)SNHG3 aggravates the osteogenic differentiation of valvular interstitial cells(VICs)in the pathogenesis and calcific aortic valve(CAVD)progression.However,the mechanism of SNHG3 promoting the osteogenic differentiation of VICs is still unknown,and its molecular mechanism needs to be further explored in order to as a nevol therapeutic target in human CAVD.Methods:Using primary human VICs to promote calcification and culture,knockdown and overexpress SNHG3,respectively,and then perform transcriptomic sequencing.Next,bioinformatics methods are used to screen common differential genes and signaling pathways.Combined with literature review and polymerase chain reaction(PCR)and western blotting were verified to screen out BMP2 protein and its signaling pathway in osteoblast differentiation of VICs.BMP2 pathway was verified by two rescue experiments.VICs supplemented BMP2(50ng/ml)on the basis of knockdown of SNHG3 and supplemented the BMP2 pathway inhibitor LDN-193189(100ng/ml)on the basis of overexpression of SNHG3 to detect expression of bone differentiation proteins(OPN,RUNX2,OCN),The spatial distribution of SNHG3 in VICs without and after calcification was determined by RNA in situ hybridization and subcellular RNA fractionation of hVICs.The mechanism that SNHG3 functions in cells was explored through bioinformatics analysis and literature review,and the promotion of aortic valve by SNHG3 was verified by RNA co-immunoprecipitation,RNA-pull down,chromatin co-immunoprecipitation,bisulfite sequencing PCR and other techniques mechanism of calcification.Results:SNHG3 knockdown and overexpressed SNHG3 in VICs were identified by transcriptomic sequencing.There were 6 differential genes that were downregulated in the SNHG3 knockdown group and upregulated in the SNHG3 overexpression group and upregulated in the SNHG3 overexpression group and downregulated in the SNHG3 knockdown group.Among them,BMP2 had the most significant difference,gene set enrichment analysis,and KEGG enrichment analysis found that the TGF-β signaling pathway was significantly up-regulated when SNHG3 was overexpressed.PCR and western blot analysis showed that the expression of BMP2 was significantly downregulated after knockdown of SNHG3;the expression of BMP2 was significantly upregulated after overexpression of SNHG3.Rescue experiments showed that supplementation of BMP2 on the basis of SNHG3 knockdown could up-regulate the expression of osteogenic differentiation proteins(OPN,RUNX2,OCN);supplementation of BMP2 pathway inhibitors on the basis of SNHG3 overexpression could inhibit the expression of osteogenic differentiation proteins.RNA in situ hybridization experiments and nuclear and cytoplasmic PCR showed that SNHG3 was mainly distributed in the nucleus,and the spatial distribution of SNHG3 did not change after calcificationpromoting culture.Bioinformatics analysis,RNA co-immunoprecipitation,RNA-pull down showed that SNHG3 binds to the core catalytic subunit EZH2 of most protein complexes,and chromatin co-immunoprecipitation,western blot and bisulfite sequencing PCR showed that SNHG3 could bind to EZH2 After inhibiting the level of H3K27m3 in the BMP2 promoter region,it promotes the expression of BMP2.Conclusion:During the osteogenic differentiation of VICs,SNHG3 binds to the core catalytic subunit EZH2 of the polycomb complex in the nucleus,inhibits the level of H3K27m3 in the BMP2 promoter region,upregulates the transcription of BMP2 and activates its pathway,and finally promotes aortic valve calcification. |
