miR-140-5p靶向转化生长因子β受体Ⅰ调控乳小鼠心肌成纤维细胞的胶原表达

miR-140-5p target transforming growth factor-β receptorⅠregulates collagen expression in mouse cardiac fibroblasts

目的探究微RNA-140-5p(miR-140-5p)对转化生长因子(TGF)β受体Ⅰ(TβRⅠ)的靶向调控作用和机制。方法①乳小鼠心肌成纤维细胞(MCF)给予高浓度葡萄糖(HG)30 mmol·L^-1或TGF-β15μg·L^-1处理24 h后,荧光定量PCR法检测miR-140-5p mRNA表达。②HEK293T细胞分为细胞对照组、转染miR-140-5p模拟物(mimics)30 nmol·L^-1组、转染含miR-140-5p结合位点的野生型TβRⅠ3'UTR序列双荧光素酶载体(WT)1 ng·L^-1组、突变型双荧光素酶载体(MT)1 ng·L^-1组、WT 1 ng·L^-1+miR-140-5p mimics30 nmol·L^-1组和MT 1 ng·L^-1+miR-140-5p mimics 30 nmol·L^-1组,48 h后采用化学发光法检测萤火虫荧光素酶和海肾荧光素酶活性。③对MCF转染miR-140-5p mimics 24 h后,给予HG 30 mmol·L^-1或TGF-β15μg·L^-1处理24 h,Western印迹法检测转染MCF中TβRⅠ,p-Smad2,Smad7,Ⅰ型胶原蛋白(CollⅠ)和CollⅢ蛋白表达水平。结果①与细胞对照组比,HG 30 mmol·L^-1和TGF-β15μg·L^-1组MCF中miR-140-5p表达显著降低(P<0.05)。②双荧光素酶报告基因实验结果显示,与细胞对照组相比,HEK293T细胞转染miR-140-5p mimics组和WT组荧光活性显著降低(P<0.05),与WT组相比,WT+miR-140-5p mimics荧光活性显著降低(P<0.05),与miR-140-5p mimics组相比,miR-140-5p mimics+WT组荧光活性显著降低,而miR-140-5p mimics+MT组荧光活性显著升高(P<0.05)。③与细胞对照组相比,转染miR-140-5p mimics组TβRⅠ,CollⅠ和CollⅢ蛋白表达水平及Smad2磷酸化水平显著降低(P<0.05),Smad7蛋白表达增加(P<0.05);与miR-140-5p mimics组相比,miR-140-5p mimics+HG 30 mmol·L^-1和miR-140-5p mimics+TGF-β15μg·L^-1组TβRⅠ,CollⅠ和CollⅢ蛋白表达及Smad2磷酸化水平显著升高(P<0.05),Smad7蛋白表达显著降低(P<0.05)。结论 miR-140-5p能靶向TβRⅠ并通过TGF-β/Smad信号通路调控HG及TGF-β1诱导的MCF胶原表达。

OBJECTIVE To explore the targeted regulatory effect of miR-140-5 p on transforming growth factor(TGF)-βreceptorⅠ(TβRⅠ)and the regulatory mechanism bewteen miR-140-5 p and TβRⅠ.METHODS①Mouse cardiac fibroblasts(MCF)were treated with glucose 30 mmol·L^-1(HG)or transforming growth factor-β1(TGF-β1)5μg·L^-1 for 24 h.The expression of miR-140-5 p was detected by qRT-PCR method.②Dual luciferase reporter gene vectors(wild and mutant-type)and miR-140-5 p mimics were transfected or co-transfected into HEK293 T cells that were divided into cell control group,transfected miR-140-5 p mimics 30 nmol·L^-1 group,transfected wild type dual luciferase reporter gene vectors(WT)of TβRⅠ3′UTR sequences containing miR-140-5 p binding sites in 1 ng·L^-1 group,mutant type dual luciferase reporter gene vectors(MT)1 ng·L^-1 group,WT 1 ng·L^-1+miR-140-5 p mimics30 nmol·L^-1 group and MT 1 ng·L^-1+miR-140-5 p mimics 30 nmol·L^-1 group.After 48 h of transfection,the activities of firefly and renilla luciferase were tested by chemiluminescence method.③The expressions of TβRⅠ,Smad7,collagen typeⅠ(CollⅠ),CollⅢand phosphorylation level of Smad2 were detected by Western blotting after transfecting miR-140-5 p mimics 24 h and treating them with HG30 mmol·L^-1 or TGF-β15μg·L^-1 in MCF.RESULTS①Compared with cell control group,the expression of miR-140-5 p in MCF decreased significantly in HG 30 mmol·L^-1 group and TGF-β15μg·L^-1 group(P<0.05).②Compared with cell control group,the fluorescence activity of reporter gene of miR-140-5 p mimics and WT groups was decreased significantly(P<0.05).Compared with WT group,the fluorescence activity of reporter gene of miR-140-5 p mimics+WT group was decreased significantly(P<0.05).Compared with miR-140-5 p mimics group,the fluorescence activity of reporter gene of miR-140-5 p mimics+WT was decreased while that of mi R-140-5 p mimics+MT group was upregulated significantly(P<0.05).③Compared with cell control group,the protein expressions of TβRⅠ,CollⅠ,CollⅢand phosphoryation level of Smad2 significantly decreased,but Smad7 protein was upregulated(P<0.05).Compared with miR-140-5 p mimics group,the protein expressions of TβRⅠ,CollⅠ,CollⅢand phosphorylation level of Smad 2 significantly increased in miR-140-5 p mimics+HG 30 mmol·L^-1 and miR-140-5 p mimics+TGF-β15μg·L^-1 groups,while the expression of Smad7 protein was significantly decreased(P<0.05).CONCLUSION miR-140-5 p can target TβRⅠand regulate collagen expression induced by HG and TGF-βvia TGF-β/Smads signaling pathway in MCF.