| BackgroundIn China,the mortality of acute myocardial infarction has been rising for the past few years,causing a high disease burden.The development and clinical application of thrombolytic therapy and cardiac interventional therapy have contributed to a much better prognosis,but there are still a lot of patients whose condition would progress to cardiac failure due to the occurrence of ventricular remodeling.Ventricular remodeling after myocardial infarction includes a plurality of processes,such as myocardial hypertrophy,extracellular matrix synthesis and deposition,angiogenesis and the like.In the initial stage of myocardial infarction,extracellular matrix synthesis and deposition gradually increased,on the one hand,it can make the heart contractility,on the other hand,scarring can protect cardiac rupture.However,in the late stage of myocardial infarction,excessive deposition of extracellular matrix can lead to myocardial fibrosis,and ultimately progress to heart failure.Many studies have found that,in the course of myocardial infarction,the infarct area will continue to appear synthesis and deposition of extracellular matrix,or even in a few years can still be observed in the active state of the extracellular matrix remodeling.Excessive myocardial fibrosis is one of the key pathological changes in ventricular remodeling,and cardic fibroblasts(CFs)play an important role in the process,whose activation is crucial to myocardial fibrosis.Numerous studies have explored the role of microRNAs(miRNAs)in various diseases.MiRNAs are a class of noncoding small RNAs highly conserved during biological evolution,with a length of about 22 nucleotides.They do not encode proteins but can play a regulatory role by binding with corresponding sites in 3' untranslated regions(3'UTR)of target genes.MiR-125b in the myocardial tissue of people with heart failure and other cardiac tissue in animal models were found upregulated,suggesting that miR-125b may be myocardial remodeling characteristic molecules.Previous studies have shown that both microRNA and Wnt/?-catenin signaling pathway participate in the regulation of cardic fibroblasts,they can be potential targets for the intervention of myocardial fibrosis.The present study conducts a series of experiments to investigate whether miR-125b can regulate cardic fibroblasts,and if the effect takes place via Wnt/p-catenin signaling pathway.The study contains three parts.Part 1Objective:To investigate whether miR-125b can regulate the synthesis,activation,proliferation,migration and apoptosis of CFs.Methods:miR-125b mimics and inhibitors were transfected into CFs.qRT-PCR was used to detect the mRNA of collagen type I,III,and Western blot was used to detect the protein expression of collagen type ?,? and a-SMA.We used BrdU incorporation assay to measure cell proliferation after transfection.Transwell migration assay was used to measure cell migration,and Flow Cytometry Method(FCM)was conducted to detect cell apoptosis.Results:1.By transfecting miR-125b mimics into CFs,we found the over expression of miR-125b could promote the synthesis of col-? and col-?(all P<0.05),while transfecting miR-125b inhibitors into CFs would inhibit the synthesis of col-1 and col-3(all P<0.05).2.After the transfection of miR-125b mimics,the over-expressed miR-125b increased the expression of a-SMA(myofibroblast transformation marker)at protein level(P<0.05),while the miR-125b inhibitors inhibited its expression(P<0.05).3.Over-expressed miR-125b promoted the proliferation level in CFs(P<0.05),while the miR-125b inhibitors inhibited CFs' proliferation(P<0.05).4.Over-expressed miR-125b promoted the migration of CFs(P<0.05),while the miR-125b inhibitors inhibited CFs'migration(P<0,05).5.The miR-125b inhibitors upregulated the apoptosis of CFs(P<0.05).Conclusion:1.miR-125b promotes the synthesis of col-I and col-? in CFs.2.miR-125b promotes CFs' differentiation into myofibroblast.3.miR-125b promotes proliferation and migration level of CFs.4.miR-125b inhibitors promotes apoptosis of CFs.Part 2Objective:To predict and verify target genes of miR-125b in the TGF-?1 or Wnt/?-catenin signaling pathway.Methods:Bioinformatics algorithm was used to predict target genes of miR-125b,and we conducted Dual luciferase assay to verify selected target gene.Results:1.MMP 14,MAPK1,SFRP5,FGFR3 and FGF15 were predicted to be the target genes of miR-125b,we selected SFRP5 to conduct further verification.2.In CFs transfected with wild type SFRP5-3'UTR,the activity of luciferase decreased by 45%(P<0.05),while there was no significant change in CFs transfected with mutant SFRP5-3'UTR.Conclusion:SFRP 5 is a target gene regulated by miR-125b.Part 3Objective:To test and verify whether miR-125b influence CFs by regulating the expression of SFRP5.And to investigate the effect of miR-125b on myocardial tissue in vivo.Methods:1.miR-125b mimics,miR-125b inhibitors,overexpression vector of SFRP5,SFRP5 siRNA,miR-125b mimics and overexpression vector of SFRP5 were transfected into CFs.qRT-PCR was used to detect the mRNA of SFRP5,and Western blot was used to detect the protein expression of SFRP5,collagen type ?,1? and a-SMA.We used BrdU incorporation assay to measure cell proliferation after transfection.Transwell migration assay was used to measure cell migration,and Flow Cytometry Method(FCM)was conducted to detect cell apoptosis.2.We built mice model of acute myocardial infarction to observe the changes of myocardial morphology by inhibiting the expression of miR-125b.And We used immunohistochemical method to detect the expression of SFRP5 in myocardial tissue to investigate the influence of miR-125b on SFRP5.Results:1.By transfecting miR-125b inhibitors into CFs,we observed the expression of SFRP5 is upregulated(P<0.05);while SFRP5 was downregulated by transfecting miR-125b mimics into CFs(P<0.05).2.By transfecting miR-125b inhibitors into CFs,we observed the expression of SFRP5 is upregulated(P<0.05);while SFRP5 was downregulated by transfecting miR-125b mimics into CFs(P<0.05);By transfecting overexpression vector of SFRP5 into CFs,the expression of SFRP5 is upregulated,the expression of col-?,? and ?-SMA(myofibroblast transformation marker)were inhibited(all P<0.05);while it showed opposite effect when SFRP5 siRNA was transfected(all P<0.05);There were no significant changes of these expressions when transfected with both overexpression vector of SFRP5 and miR-125b mimics.3.SFRP5 siRNA inhibited the expression of SFRP5,thus promoted the proliferation of CFs(P<0.05);while overexpression vector of SFRP5 inhibited the proliferation(P<0.05);There was no significant change of CFs' proliferation when transfected with both overexpression vector of SFRP5 and miR-125b mimics.4.SFRP5 siRNA promoted the migration of CFs(P<0.05);while overexpression vector of SFRP5 inhibited the migration(P<0.05);There was no significant change of CFs' migration when transfected with both overexpression vector of SFRP5 and miR-125b mimics.5.SFRP5 siRNA protected CFs from apoptosis;while overexpression vector of SFRP5 facilitated the process(P<0.05);There was no significant change of CFs' apoptosis when transfected with both overexpression vector of SFRP5 and miR-125b mimics.6.HE staining,sham operation group showed myocardial stripes clear,neat,clear nucleus,no cell swelling;acute myocardial infarction group showed more divergence in necrosis,clear boundaries,with a clear fibroblast proliferation,cardiac hypertrophy,more fibrosis;after transfection into miR-125b inhibitors,acute myocardial infarction typical pathological phenomenon significantly improved.7.Masson staining showed that in the sham operation group,the myocardial tissue was stained evenly red,showing the thickness of the anterior wall is more consistent,less collagen fibers in the myocardium,no cord and fusion collagen fibers;In the acute myocardial infarction group,the myocardium of the left ventricular anterior myocardial infarction area was replaced by collagen fibers,which was increased in the basal ganglia;In the acute myocardial infarction group+ miR-125b inhibitor transfection group,the typical pathological phenomenon was improved after acute myocardial infarction.The results of Masson staining were analyzed by IPP6.0 and the volume fraction of collagen was calculated,The collagen volume fraction in acute myocardial infarction group was significantly higher than that in sham operation group(P<0.05).The volume fraction of collagen in the acute myocardial infarction + miR-125b inhibitor transfection group was significantly lower than that in the acute myocardial infarction group.8.Immunohistochemistry assay showed SFRP5 was down-regulated after AMI(P<0.05),and the expression of SFRP5 in myocardial tissue of acute myocardial infarction + miR-125b inhibitor group was significantly higher than that in acute myocardial infarction group(P<0.05).Conclusion:1.SFRP5 inhibits cell synthesis of col-? and col-?,differentiation,proliferation and migration of CFs,but,SFRP5 promotes apoptosis of CFs.2.miR-125b increases cell synthesis,differentiation,proliferation and migration of CFs via inhibiting the expression of SFRP5.3.Myocardial morphology after AMI can be improved by inhibition of the expression of miR-125b.Total Conclusion:1.miR-125b increases cell synthesis of col-? and col-?,differentiation,proliferation and migration of CFs,and Inhibition of miR-125b promotes apoptosis of CFs.2.SFRP5 inhibits cell synthesis of col-I and col-?,differentiation,proliferation and migration of CFs,but SFRP5 promotes apoptosis of CFs.3.miR-125b regulates function and activity of CFs via inhibiting the expression of SFRP5.4.Myocardial morphology after AMI can be improved by inhibition of the expression of miR-125b. |
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