Construction And Screening Of Long Noncoding RNA Expression Library In Experimental Autoimmune Myocarditis

Part 1 Construction of dynamic lncRNA-gene expression library for experimental autoimmune myocarditisObjective: To immunize mice with purified cardiomyosin to establish experimental autoimmune myocarditis(EAM)model,and use lncRNA gene chip technology to construct lncRNA-gene expression library for experimental autoimmune myocarditis.Methods: Male BALB / c mice of 6-8 weeks were selected as the research object,and EAM models were established by immunizing myocardial myosin heavy chain.All mice were randomly quadripartite: normal control group(EAM day 0),EAM day 14 group,EAM day 21 group and EAM day 62 group.Measure and record the body weight of experimental mice.Hearts were obtained on days 14,21,and 62 of EAM model mice,and heart weight / body weight ratio(HW / BW)was calculated and recorded.Hematoxylin-eosin staining(HE staining)was used to detect the inflammation and infiltration of mouse heart tissue,and Masson staining was used to detect the degree of fibrosis in mouse heart tissue.lncRNA gene chip technology was used to detect abnormal expression of lncRNA and gene in myocardial tissues on the 14 th,21st and 62 nd days of the EAM model.Results: Compared with the normal control group(Day 0 of EAM),EAM model mice showed inflammation and infiltration of myocardial tissue on day 14;the inflammation of myocardial tissue peaked on day 21 and myocardial fibrosis was observed;on day 62 Significant fibrosis occurs and the ventricles expand.In the constructed EAM model lncRNA-gene expression library,there are 1191 and 2121 differentially expressed lncRNA and gene,respectively.The differentially expressed lncRNA and gene are mainly distributed on chromosomes 1-19,and are visible in sex chromosomes X and Y.The top 20 lncRNAs with high expression of the lncRNA-gene expression library on Days 14,21 and 62 are as follows: 1.Day 14: NONMMUT011100,NR?038116,NONMMUT009926,NR?045561,XR?105914,NONMMUT072700,NONMMUT016470,ENSMUST00000120641,NONMMUT052111,NONMMUT062616,NONMMUT003874,ENSMUST00000131638,ENSMUST00000156603,uc029 tti.1,uc029 tto.1,uc029 ttw.1,uc029 tup.1,NONMMUT015496,NONMMUT034142,NONMMUT037710,NONMMUT063122,NONMMUT002947;2.Day 21: XR?105914,NONMMUT074550,ENSMUST 141,NRSMUT00000 NONMMUT072700,NONMMUT001509,NONMMUT071550,XR?140674,NONMMUT000091,NONMMUT015496,NONMMUT036082,NR?104383,NONMMUT048938,OTTMUST00000044377,KnowTID₀0002271,NONMMUT055574,ENSMUST00000122415,NONMMUT062616;3.day 62: NONMMUT036082,KnowTID₀0002271,NONMMUT048938,NONMMUT003003,NR?028478,NONMMUT003096,KnowTID₀0006493,NONMMUT028345,ENSMUST00000083883,KnowTID₀0006395,NR?002842,KnowTID₀0003470,NONMMUT 022746,NONMMUT062809,NONMMUT048006,uc029 xia.1,uc029 xmk.1,uc029 roz.1,ENSMUST00000083102,NONMMUT068517.Conclusions:By constructing the EAM model to simulate the acute and chronic phases of myocarditis,we constructed the EAM model lncRNA-gene expression library and found abnormally expressed lncRNA and gene,which laid the foundation for further analysis of the abnormally expressed lncRNA function.Part 2 Screening and bioinformatics analysis of differential lncRNAs in lncRNA-gene expression libraryObjective: By analyzing the EAM model lncRNA-gene expression library constructed in the previous part of the experiment,to obtain functional lncRNA and corresponding genes.Methods: The EAM model was constructed,and the myocardial tissues of mice were obtained on day 14,21 and 62.Through lncRNA-gene co-expression net-work analysis to find differential genes co-expressed by lncRNA.Through the analysis of gene expression time trend,find the lncRNA and gene with the same expression trend in the expression library.Study the potential function of lncRNA through GO analysis.Pathway analysis was used to study the signaling pathways involved in lncRNA.The expression of lncRNA was verified by real-time fluorescence quantitative PCR.Results: We screened the top 5 lncRNAs that were highly expressed on the 14 th,21st,and 62 th days of the EAM model: NR045561,ENSMUST00000120641,NONMMUT062616,NONMMUT015496,NR104383,and verified these 5 lncRNAs by real-time quantitative PCR Expression in myocardial tissue.Co-expression analysis found that lncRNA NONMMUT062616 had no corresponding co-expressed genes,lncRNA NR?045561 had 55 co-expressed genes,lncRNA ENSMUST00000120641 had 49 co-expressed genes,lncRNA NONMMUT015496 had 11 co-expressed genes,and lncRNA NR?104383 had 82 co-expressed genes.Significant analysis of the gene expression trend found that the lncRNA NR045561,ENSMUST00000120641,NONMMUT015496,NR104383 and corresponding co-expressed genes all fell in the time trend 21(expression was up-regulated and then entered the plateau period,and continued to be down-regulated after a period of time,which is consistent with the acute phase of the EAM model.The inflammatory changes that progress in the chronic phase are the same).GO analysis and Pathway analysis found that lncRNA NR045561,ENSMUST00000120641,NONMMUT015496,and NR104383 were involved in inflammation-related signaling pathways,lncRNA NR?045561 was mainly involved in the chemotaxis and activation of neutrophils,and lncRNA ENSMUST00000120641 was mainly involved in the chemotaxis of monocytes / macrophages.Cardiac fibroblast activation,lncRNA NONMMUT015496 is mainly involved in the chemotaxis of T cells and manages the production of vascular endothelial growth factor,while lncRNA NR?104383 is mainly involved in the chemotaxis of T cells,the differentiation of killer toxic T cells and the activation of B cells.Conclusions:In the EAM model lncRNA-gene expression library,lncRNA NR045561,ENSMUST00000120641,NONMMUT062616,NONMMUT015496,NR104383 are highly expressed.Functional analysis found that lncRNA NR045561,ENSMUST00000120641,NONMMUT015496,NR104383 are all involved in the activation and chemotaxis of inflammatory cells in the inflammatory response.Part 3 Study on the potential mechanism of lncRNA ENSMUST00000120641 in experimental autoimmune myocarditisObjective: To explore the potential mechanism of lncRNA ENSMUST00000120641 in experimental autoimmune myocarditis(EAM).Methods: EAM model was constructed to obtain mouse myocardial tissue on day 21.The expression of lncRNA ENSMUST00000120641,Ccr2 and Timp1 was detected by real-time fluorescence quantitative PCR.The mononuclear cells of heart,spleen and peripheral blood were isolated,and the expressions of lncRNA ENSMUST00000120641 and Ccr2(chemokine(C-C motif)receptor 2)were detected by real-time fluorescence quantitative PCR.The expression of Ccr2 and Timp1(TIMP Metallopeptidase Inhibitor 1)in myocardial tissue was detected by Western blot.The expression and subcellular localization of lncRNA ENSMUST00000120641 in myocardial tissue were detected by FISH.The transcription factors of Ccr2 and Timp1 were analyzed by PROMO database.The binding relationship between lncRNA ENSMUST00000120641 and transcription factors of Ccr2 and Timp1 was analyzed by RIPSeq database.Results: The EAM model mice had ventricular cavity enlargement on the 21 st day,decreased ejection fraction,and decreased cardiac function.lncRNA ENSMUST00000120641 is highly expressed in myocardial tissue and is mainly distributed in the nucleus.lncRNA ENSMUST00000120641 and Ccr2 are also highly expressed in heart,spleen and peripheral blood mononuclear cells.Ccr2 and Timp1 are expressed in myocardial tissue at both mRNA and protein levels.PROMO database found that Ccr2 has 11 closely related transcription factors: C/EBPbeta,YY1,c-Jun,HOXAS,c-Fos,C/EBPalpha,MyoD,NF-KappaB,HES-1,JunD,NF-AT4;Timp1 There are 10 closely related transcription factors: C/EBPbeta,c-Fos,NF-1,HOXAS,HES-1,AP-1,JunD,c-Jun,MyoD,C/EBPbeta.By analyzing the binding relationship between lncRNA ENSMUST00000120641 and these transcription factors,it was found that lncRNA ENSMUST00000120641 had a close binding relationship with the transcription factor NF-KappaB(nuclear factor of kappa light polypeptide gene enhancer in B cells 1)predicted by Ccr2,and a close relationship with the transcription factor NF-1 predicted by Timp1.Conclusion: lncRNA ENSMUST00000120641 may regulate the expression of Ccr2 by combining with NFkappa B and exert its effect on the chemotaxis of monocytes / macrophages in the EAM model;lncRNA ENSMUST00000120641 may play a role in influencing myocardial fibrosis in the EAM model by regulating the expression of Timp1 by binding to NF-1.