MiR-30a-5p Supresses Cardiac Fibrosis Through TGF-β1 Pathway By Targeting ITGB3

Aims: MicroRNAs(miRNAs)are confirmed their crucial role in myocardical infarction.MiRNA-30a-5p,a member of the miR-30 family,has been implicated in the TGF-β1 pathway in fibrosis after myocardial infarction.Integrin β3 is reported to express on platelets and endothelial cell and to influence the expression of chemokines and cytokines,including transforming growth factor β1(TGF-β1).The present study aimed to observe the role of miR-30 a in regulation of Integrin β3 and to explore the expression between miR-30 a 、 Integrin β3 and TGF-β1 in myocardial infarction.Methods:1.We identified integrin b3 as a putative target of miR-30 a and an upstream regulator of TGF-β1 from some online software tools,including TargetScan version6.2(http://www.targetscan.org),MiRanda(http://www.microrna.org),DIANAmicroT version 3.0(http://diana.cslab.ece.ntua.gr,)and miRDB(http://mirdb.org/miRDB).2.Cardiac fibroblasts were isolated from the hearts of 1-3-day-old Sprague-Dawley rat pups.The fibroblats were divided into 4 group(each group included 2dishes).Three of the 4 groups were respectively infected with purified viruses,including LV-rno-miR-30a-5p agonist(precursor-rno-miR-30a;Shanghai Genechem Co.,Ltd.,Shanghai,China)、 and LV-rno-miR-30a-5p-inhibition(precursor-rno-miR-30a-inhibition;Shanghai Genechem Co.,Ltd.,Shanghai,China)and negative control vector(NC)(hu6-MCS-Ubiquitin-EGFP-IRES-puromycin;Shanghai Genechem Co.,Ltd.)and another groups was cultured with no-virusDulbecco’s modified Eagle’s medium(DMEM)with 10% fetal bovine serum(FBS)and penicillin/streptomyc.For oxygen-glucose deprivation-treated cells(1 dish of each group),Cardiac fibroblasts were incubated with DMEM without glucose in a humidified atmo-sphere containing 5% CO2 and 95% N2(v/v)at 37℃ for 6 h,then the culture medium was removed,and fresh DMEM containing 10% FBS was added to dishes in a regular 5% CO2 incubator.We setted 5group,including sham,sham+hypoxia,hypoxia+miRNA-30a-agonist,hypoxia+miRNA-30a-inhibition and hypoxia+NC.The mRNA level of miRNA-30 a,TGF-β1 and Integrin β3 were detected by realtime-quantitative PCR analysis(rt-PCR).The protein level of TGF-β1and Integrin β3 were detected by Western Blot analysis(WB).3.The cells were divided into four dishes.Two dishes were infected with LV-rno-miR-30a-5p-agonist.One dish and one virus-treated dish were transfected with specific siRNA for silencing Integrin β3 expression.These 4 dishes were cultured in DMEM without glucose in a humidified atmo-sphere containing 5% CO2 and95% N2(v/v)at 37℃ for 6 h,then the culture medium was removed,and fresh DMEM containing 10% FBS was added to dishes in a regular 5% CO2 incubator.Then the expression of TGF-β1 and intergin β3 were detected by rt-PCR and WB.Results:1.The expressions of TGF-b1 and the ITGB3 were increased in hypoxia atmosphere.The result of the rt-PCR indicated that tthe TGF-b1 mRNA levels and the ITGB3 mRNA levels were increased in hypoxia+miRNA-30a-inhibition group and decreased in hypoxia+miRNA-30a-agonist group,comparing to the hypoxia+NC group,(P<0.05).And the protein level of TGF-β1 and integrin β3 were the same trend with rt-PCR,(P<0.05).Hypoxia leaded to increased expression of integrin β3 and TGF-β1,(P<0.05).2.By interfering siRNA to slience the expression of integrin β3,the expression of TGF-β1 was obviously increased in the interference group comparing to the noninterference group,(P<0.05).Conclusion:Our results suggest that miR-30a-5p can regulate the expression of TGF-β1 through the inhibition of integrin β3 expression in rats.Our results also indicate that miRNA-30a-5p is as a probable index in the identification of myocardial ischemia after acute myocardial infarction.