| Myocardial infarction(MI)is a common cardiovascular disease and the main factor of heart failure(HF),with high morbidity and mortality.After MI,cardiac fibroblasts(CFs)are activated,and then proliferate,synthesize and secrete collagens.These collagen proteins form extracellular matrix(ECM)to maintain the integrity of the heart structure and avoid the injury of left ventricular.Nevertheless,the excessive proliferation of CFs can cause overformation,secretion and deposition of collagens,which induce the myocardial fibrosis,and thereby leading to myocardial elastic damage and heart failure.Although the roles of the proliferation and collagen formation in MI induced myocardial fibrosis has been widely studied,the underlying mechanism has not been fully elucidated.Tumor protein p53 induced nuclear protein 1(TP53INP1)is a pressure-induced gene,which can inhibit the proliferation and collagen formation of CFs.c-Myc is a proto oncogene,which not only participates in the occurrence and development of various cancers,but also plays an important regulatory role in cardiovascular diseases such as ischemic cardiomyopathy,cardiac hypertrophy and diabetic cardiomyopathy.In addition,it has been confirmed that c-Myc can inhibit the expression of TP53INP1 in esophageal cancer.However,it is not clear whether c-Myc can regulate the proliferation and collagen formation of CFs through TP53INP1 to influence the progress of myocardial fibrosis.Micro RNA(miRNA)is a kind of non coding RNA.In recent years,it has been found that miRNA is also widely involved in the pathological process of many cardiovascular diseases.In this study,we will establish a mouse myocardial fibrosis model(in vivo)induced by MI and myocardial fibrosis model(in vitro)induced by transforming growth factor beta 1(TGF-?1)or angiotensin II(Ang II).HE staining,immunohistochemical staining,cell transfection,overexpression,silence technology,luciferase reporter gene,MTT experiments,real-time quantitative PCR(qRT-PCR),and western blot assays were utilized to investigate the function and mechanism of miR-142-5p and miR-212-5p in myocardial fibrosis of MI mice.Our study might accumulate more data for the pathological mechanism of myocardial fibrosis after MI and provide effective molecular targets for the diagnosis and treatment of myocardial fibrosis.Part one The expression of miR-142-5p and miR-212-5p expressions are down-regulated in myocardial fibrosisObjective: miR-142-5p and miR-212-5p are involved in the development of cardiovascular diseases.The purpose of this study was to investigate the expression of miR-142-5p and miR-212-5p in myocardial fibrosis models in vitro and in vivo.Methods: MI-induced myocardial fibrosis model was established by ligation of the left anterior descending branch of coronary artery.Primary CFs were isolated from neonatal mice and induced by TGF-? 1 and Ang II to establish myocardial fibrosis model in vitro.The left ventricular ejection fraction(LVEF)and fraction shortening(LVFS)were detected by echocardiogram.Myocardial histopathology was observed by HE staining.Myocardial fibrosis was evaluated by the immunohistochemical staining of Collagen I and Collagen III.The expressions of Collagen I,Collagen III,miR-142-5p and miR-212-5p were detected by qRT-PCR and western blot.Result:1.The results of echocardiogram showed significant decreased LVEF and LVFS of MI mice.2.HE staining showed loose and disordered structure of myocardial fibers,large distribution area of collagen,and denatured or broken myocardial cells of MI mice.3.Immunohistochemistry and western blot showed increased expression of Collagen I and Collagen III in the myocardial tissue of MI mice.4.The expressions of miR-142-5p and miR-212-5p in the myocardial tissue of mice were down-regulated 7,14 and 28 days after the MI model established.5.The expressions of miR-142-5p and miR-212-5p were decreased in TGF-?1-and Ang II-induced CFs.Conclusion:1.The cardiac function of MI mice was damaged and myocardial fibrosis occurred.2.miR-142-5p and miR-212-5p were down-regulated in the in vivo and in vitro model of myocardial fibrosis.Part two miR-142-5p and miR-212-5p inhibits the proliferation and collagen formation of mouse cardiac fibroblastsObjective: The proliferation and collagen formation of CFs are important processes of myocardial fibrosis.The purpose of this study was to investigate whether miR-142-5p and miR-212-5p can affect the proliferation and collagen formation of CFs.Methods: Primary CFs were isolated from neonatal mice and transfected with the overexpression vectors of miR-142-5p and miR-212-5p.Then CFs were induced by TGF-?1 and Ang II to establish myocardial fibrosis model in vitro.The proliferation of CFs was detected by MTT assay and the protein levels of Collagen I and Collagen III in CFs were detected by western blot.Result:1.Overexpressing miR-142-5p or miR-212-5p alone or simultaneously inhibited the proliferation of TGF-?1-and Ang II-induced CFs cells.2.Overexpressing miR-142-5p or miR-212-5p alone or simultaneously decreased the expression of Collagen I and Collagen III in TGF-?1-and Ang II-induced CFs.Conclusion:miR-142-5p and miR-212-5p cooperatively inhibited the proliferation and collagen formation of TGF-?1-and Ang II-induced CFs cells CFs.Part three miR-142-5p and miR-212-5p inhibits the proliferation and collagen formation of cardiac fibroblasts through c-Myc/TP53INP1 pathwayObjective: TP53INP1 can inhibit the proliferation and collagen formation of CFs,and c-myc can inhibit the expression of TP53INP1.The aim of this study was to explore whether miR-142-5p and miR-212-5p regulate the expression of TP53INP1 by targeting c-Myc,thereby affecting the proliferation and collagen formation of CFs.Methods: Bioinformatics software(Target Scan)was used to predict the miR-142-5p and miR-212-5p binding sites in the 3'-untranslated region(3'-UTR)of c-Myc.Primary CFs were isolated from neonatal mice and transfected with overexpression vectors of miR-142-5p,miR-212-5p or c-Myc or silence vectors of miR-142-5p and miR-212-5p in CFS cells.Then CFs were induced by TGF-?1 and Ang II to establish myocardial fibrosis model in vitro.The relationship between c-Myc and those two miRNAs were confirmed by co-transfection technology and luciferase reporter gene experiment.The proliferation of CFs was detected by MTT assay.The m RNA expression level of c-Myc was determined by qRT-PCR.The protein levels of Collagen I,Collagen III,c-Myc and TP53INP1 were measured by western blot.Result:1.Bioinformatics analysis showed that miR-142-5p and miR-212-5p were both located on chromosome 11 of mice,and there were miR-142-5p and miR-212-5p binding sites on c-myc 3 '-UTR.2.The results of luciferase reporter gene assay showed that the overexpression of miR-142-5p and miR-212-5p alone or simultaneously significantly inhibited the luciferase activity of wild type c-Myc 3'-UTR.When the binding site of miR-142-5p was mutated,overexpressing miR-212-5p inhibited the luciferase activity of c-Myc 3'-UTR.When the binding site of miR-212-5p was mutated,overexpressing miR-142-5p inhibited the luciferase activity of c-Myc 3'-UTR.When the two binding sites of the two miRNAs were both mutated,the overexpression of miR-142-5p or/ and miR-212-5p had no significant effect on the luciferase activity of c-Myc 3'-UTR.3.Overexpression of miR-142-5p and miR-212-5p decreased the m RNA and protein levels of c-Myc in TGF-?1-and Ang II-induced CFs.4.Silence of miR-142-5p and miR-212-5p increased the m RNA and protein levels of c-Myc in CFs.5.Overexpression of miR-142-5p and miR-212-5p promoted the expression of TP53INP1 in TGF-?1-and Ang II-induced CFs,and the effect was reversed by c-Myc overexpression.6.Overexpression of miR-142-5p and miR-212-5p inhibited the proliferation of TGF-?1-and Ang II-induced CFs,while this effect was abolished by c-Myc overexpression.7.Overexpression of miR-142-5p and miR-212-5p inhibited the expression of Collagen I and Collagen III in TGF-?1 and Ang II-induced CFs,and c-Myc overexpression reversed this impact.Conclusion:1.miR-142-5p and miR-212-5p target c-Myc.2.miR-142-5p and miR-212-5p negatively regulate the expression of c-Myc in CFs.3.miR-142-5p and miR-212-5p inhibited c-myc/ TP53INP1 pathway.4.miR-142-5p and miR-212-5p inhibited the proliferation and collagen formation of TGF-?1-and Ang II-induced CFs through c-Myc /TP53INP1 pathway.Part four Overexpression of miR-142-5p and miR-212-5p alleviates myocardial fibrosis of mice with myocardial infarctionObjective: The present study was aimed to investigate whether miR-142-5p and miR-212-5p regulate the progression of MI induced myocardial fibrosis of mice.Methods: The model of MI induced myocardial fibrosis was established by ligation of the left anterior descending branch of the coronary artery.The lentivirus recombinant vectors expressing miR-142-5p and miR-212-5p(LV-miR-142-5p and LV-miR-212-5p)were injected into the left ventricle of MI mice.The LVEF and LVFS of mice were detected by echocardiogram.The expressions of miR-142-5p and miR-212-5p were detected by qRT-PCR.The protein levels of Collagen I,Collagen III,c-myc and TP53INP1 were detected by western blot.Result:1.Overexpression of miR-142-5p and miR-212-5p increased LVEF and LVFS of MI mice.2.Overexpression of miR-142-5p and miR-212-5p decreased the protein levels of Collagen I,Collagen III and c-Myc,while increased the protein level of TP53INP1.Conclusion:Overexpression of miR-142-5p and miR-212-5p attenuated the MI induced myocardial fibrosis of mice. |
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