| BackgroundMyocardial fibrosis(MF)is the familiar pathology stage of a variety of cardiovascular diseases,which also is found to be one of the major form of myocardial remodeling.The mechanism of MF is complicated and unclear so far,which may be affected by RAAS system,immune system and other cytokines.The mechanism of different kinds of MF is dfferent.Many factors including Angiotensin,endothelin,NO.TGF,CTGF and calcium ion affect the occurance of MF.In general,the imbalance of synthesis,metabolism and degradation of extracellular matrix(ECM)is the major reason of MF.Cardiac fibroblasts(CFs)are the main cells that secretes ECM.They have a strong proliferation and less contraction ability.mainly responsible for the production and precipitation of ECM.The CFs can regulate the produce of collagen,the shape,structure and function of cadiocyte by the synthesis of biologically active substances.At the same time,it can interact with the cadiocytes and endothelial cells,regulate the electrical mechanical ability.the secretion of cytokines and the angiogenesis.The compensatory mechanism of myocardial injury,CFs tissue be repaired by transform,proliferation and collagen secretion.the progressive development of MF will lead to ventricular remodeling,resutlt to cardiac dysfunction.A lot of researches have confirmed that the important mechanisms of myocardial fibrosis is the transformation of CFs to myofibroblasts(MFB).MicroRNAs(miRNAs)are non-coding small RNA transcribed fron organisms own genome,which particate in life process and disease development by regulating gene expression of transcription and/or translation.The cardiovascular system owns a series of special niRNA.Studies have found that miRNAs are closely related to the pathophysiology of myocardial fibrosis,crdiac hypertrophy in cardiovascular diseases.The function of miRNAs has not been cleared by now.the research about miRNA and cardiovascular system(CVS)is still in initial stage and most the results are based on animal experiments.However,some researches pointed out that the regulating role of miRNAs might close to the Wnt signal pathway.Wnt signaling is a significant pathway in terms of governing the development process of cells and organs,especially to the variation and differentiation of stem cells.Wnt signaling pathway is a complicated protein network system.Some reports have indicated that the fibrotic lesions,fibrotic tissues and wound healing could have an important relationship with the Wnt singal pathway.Classic road and nonclassical road composes to the Wnt signaling pathway.?-catenin is one of the most important factors.The Wnt pathway was influenced by many factors such as SPRP family,WIF-1 and DKKs.It has been found that Wnt is possible to regulate the occurrence of fibrosis and the activation of effector cells,but the specific mechanism is not clear.Previous studies have indicated that miRNA and Wnt signal pathway may regulate and control the myocardial fibrosis.However,at present,the role of Wnt and miRNA is not clear,and the relationship between them in myocardial fibrosis has yet to be further studied.miR-154,located in the chromosome 14q32,usually was found in tumor tissues.The report about the relationship of miR-154 and fibrosis disease was only limted to pulmonary fibrosis.miR-154 and MFB is almost not reported.However,some studies have shown that abnormal expression of a series of microRNAs.such as miR-154.can lead to myocardial hypertrophy and heart failure.DKK(Dickkopf)is a kind of progressive conservative small gene family,is a secreted glycoprotein and the classical Wnt pathway inhibitor.Some researches found that miR-154 might regulate the Wnt signial pathway by targarting the DKK2?SFRP4?WIF-1.Therefore,it is necessary for us to explore the mechanism between miR-154 and Wnt signial pathway.Objectives To explore the function of miR-154 on myocardial fibrosis.and further clarifythe regulation mechanism of miR-154 and Wnt pathway,so to provide a new target for the treatment of myocardial fibrosis.Methods1.In the present research we use the Human Cardiac Fibroblasts-adult(HCF-a)and/or Human Cardiac Fibroblasts-fetal atrial(HCF-fa)afforded by ScienCell laboratory.After thawing and recovery,CFs were cultured and passaged in vitro,type? collagen immunofluorescence assay were employed to identify the CFs.miR-154 mimics and inhibitors(in vitro)were transfected to CFs,the BrdUmethod was used to observe the proliferation of CFs.Western blot analysis revealed alpha-smooth muscle actin(a-SMA),collagen ? and collagen ? protein expression.2.Integrating bioinformatics and literature searching to look for clues that miR-154 targets on Wnt/?-catenin signaling.Three online soft wares for bioinformatics analysis,i.e.,miRBase?miRanda and TargetScan,were used for searching miRNAs which might target on Wnt/?-catenin signaling,and for genes which gene may be miR-154 potential target genes.The full-length DKK2 3'UTR was amplified from cDNA,and then it was used to generate mutant-type DKK2 3'UTR in which the seven mutated nucleotides were underlined within the seed region of miR-154 binding site by PCR-based mutagenesis method.Both PCR fragments were cloned into psiCHECK-2 vector(Promega).Reporter vector psiCHECK-2 carrying the 3'UTR sequences of DKK2 were assayed for luciferase expression using the Dual Glo Luciferase Assay System(Promega)following the manufacturer's instructions.The experiment was performed in duplicate in three independent experiments.To further explore the function of miR-154 on MFB.DKK2 over-expression vector was constructed and DKK2 siRNA was synthetized.The experiment groups were seven:Blank control group,negative control group,miR-154 mimics transfection group.miR-154 inhibitor transfection group.DKK2 over-expression vector transfection group,DKK2 siRNA transfection group,MiR-154 mimics and DKK2 over-expression vector co-transfection group.Western blot method was used to observed the expression of DKK2??-catenin?a-SMA?Collagen ? and ?.The BrdU method was used to observe the proliferation of CFs.Cell cycle changes and apoptosis were analyzed by flow cytometry using PI staining.Examination of migration ability was by Transwell migration assay.Results1.After stimulated with miR-154 mimics or inhibitors,MTT analysis found that the proliferation of miR-154 mimics group was obviously higher than the control group(P<0.01).Western blot analysis indicated the expression of ?-SMA?collagen-? and Collagen-? also was higher than the control group(P<0.05).2.By searching miRBase?miRanda and TargetScan Gene database,and according to the relative articles,we found that miR-154 regulated Wnt/?-catenin pathway by targeting on DKK2.Further luciferase activity assay showed that exogenous expression of the wild-type DKK2 3'UTR resulted in a significant decrease in luciferase activity(P<0.01).In contrast,luciferase activity remained largely unchanged following expression of the mutant DKK2 3'UTR.Taken together,these results indicate that DKK2 is a direct target of miR-154,and miR-154 can inhibit expression of DKK2 in CFs.3.After CFs stimulated with miR-154,we found that the expression of ?-catenin??-SMA?collagen-? and Collagen-? were improved(P<0.01).The transfection of DKK2 siRNA had the same effect with the miR-154 mimics transfection group(P<0.01).4.BrdU analysis found that the proliferation of miR-154 mimics group and DKK2 siRNA group was obviously higher than other groups(P<0.01).5.Flow cytometry method showed that the proportion of Sub-G1 in miR-154 inhibitors group or DKK2 over-expression group was ascending,accelerating the apoptosis of CFs.6.Examination of migration ability,we found that miR-154 enhanced CFs migration(P<0.01),but DKK2 had the opposite effect(P<0.01).Conclusions1.miR-154 affects the activation of CFs,and promotes CF proliferation.2.miR-154 induces CFs to convert into MFB by targeting DKK2.3.miR-154 activated the Wnt signal pathway by targeting DKK2 to accelerate the proliferation and migration of CFs. |
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