PTBP1 Mediated The Effect Of LncRNA RP11-879F14.2 To Inhibit Myocardial Fibrosis By Targeting Dkk2

As the important indicator of the cardiac remodeling,myocardial fibrosis is the basic pathological process of various cardiovascular diseases.Under the stimulation of pathological factors,the proliferation and migration ability of myocardial fibroblasts are enhanced,and they are transformed into muscle fibroblasts and have stronger ability to secrete extracellular matrix.The occurrence of myocardial fibrosis can increase the hardness of the heart wall,reduce the elasticity,damage the systolic and diastolic function of the heart,and eventually develop into heart failure,which may finally may develop into heart failure.Therefore,it is necessary to explore the regulatory mechanism of myocardial fibrosis and identify effective targets for the treatment of myocardial fibrosis.In recent years,it has reported that long non-coding RNAs(lncRNAs)are involved in the regulation of myocardial fibrosis.Different from m RNA,lncRNAs don’t have the ability to encode protein because of the lack of open reading frame,and their length is generally more than 200 nucleotides.The biological functions of lncRNAs are complex and diverse,which may regulate gene expression in various ways,and are closely related to the occurrence and development of various diseases,including myocardial fibrosis.Lnc RNAs may be used as a target for the treatment of myocardial fibrosis in the future.At present,the specific mechanism of lncRNAs regulate myocardial fibrosis is still unclear.Here,we studied the function and regulation mechanism of lncRNA RP11-879F14.2 in myocardial fibrosis.Objective: To explore the regulatory mechanism of lncRNA RP11-879F14.2 involved in myocardial fibrosis.Methods: 1.Masson’s trichrome staining was performed to detect the changes of myocardial collagen in healthy controls and patients with heart failure;2.Lnc RNAs microarray was used to detect the expression of lncRNAs in the myocardium of patients with heart failure and healthy controls,RT-q PCR was performed to verify the expression of RP11-879F14.2 in myocardium of patients with heart failure and human atrial myofibroblasts treated with angiotensin Ⅱ(AngⅡ);3.Detection of the nucleocytoplasmic distribution of RP11-879F14.2by RT-q PCR and fluorescence in situ hybridization(FISH)in myocardial fibroblasts;4.Constructed the recombinant adenovirus RP11-879F14.2(r Ad-RP11-879F14.2),and infected HAFs and primary fibroblasts of neonatal mice(m CFs)with r Ad-RP11-879 F14.2,and detect the expression of fibrosis-related genes;5.The effect of lncRNA RP11-879F14.2overexpression on the proliferation and migration of fibroblasts in m CFs was detected by flow cytometry,Ed U and Trans-well assay;6.The binding of RP11-879F14.2 to polypyrimidine track binding protein 1(PTBP1)was identified based on bioinformatics prediction and double luciferase reporter gene experiment;7.Overexpress and knockdown PTBP1 in m CFs to detect the changes of fibrosis-related genes at RNA and protein levels;8.the effect of knockdown of PTBP1 on the expression of myocardial fibrosis-related genes regulated by RP11-879F14.2 was detected;9.Based on the results of RNA expression profile analysis,the target gene Dkk2 of PTBP1 was detected by RT-q PCR;10.To detect the effect of DKK2 knockdown on fibrosis related genes in m CFs;11.The m CFs were treated with actinomycin D,and the effects of RP11-879F14.2 and PTBP1 on the stability of Dkkk2 m RNA were detected by RT-q PCR.Results: 1.Masson staining results showed that myocardial fibrosis occurred in patients with heart failure;2.RT-q PCR results confirmed that RP11-879F14.2 expression increased in myocardial tissue and Ang Ⅱ induced HAFs of patients with heart failure;3.Overexpression of RP11-879F14.2 significantly inhibited the expression of myocardial fibrosis-related genes at RNA and protein levels,and inhibited the proliferation and migration of fibroblasts;4.The results of nuclear RNA component separation with RT-q PCR and FISH experiment confirmed that RP11-879F14.2 was mainly distributed in the nucleus of myocardial fibroblasts;5.Dual luciferase reporter assay confirmed RP11-879F14.2 can bind with PTBP1 and promote the expression of PTBP1 in m CFs;6.Overexpression of Ptbp1 can significantly inhibit the expression of myocardial fibrosis related genes,and knockdown of PTBP1 can reverse the inhibition of RP11-879F14.2 on the expression of fibrosis related genes in m CFs;7.The level of Dkk2 is up-regulated by overexpression of RP11-879F14.2 and PTBP1,and knockdown of DKK2 promotes the expression of fibrosis related genes.8.RP11-879F14.2 and PTBP1 could improve the stability of Dkk2 m RNA.Conclusion: Lnc RNA RP11-879F14.2 inhibits the expression of myocardial fibrosis related genes by binding to PTBP1 to enhance the expression of DKK2.