肌球蛋白重链7基因来源的miR-208b-3p促进心肌成纤维细胞中纤维化相关基因表达

Myosin Heavy Chain 7 Gene-derived miRNA-208b-3p Enhances the Fibrosis-related Gene Expression in Cardiac Fibroblasts

【目的】探究肌球蛋白重链7基因来源的微小RNA-208b-3p(miR-208b-3p)对心肌成纤维细胞纤维化表型的调控作用。【方法】通过miRNA表达谱芯片分析18周龄的糖尿病db/db小鼠和db/m对照小鼠心肌中差异的miRNAs。通过实时荧光定量PCR(RT-qPCR)检测miR-208b-3p在血管紧张素Ⅱ(AngⅡ)和高糖/葡萄糖氧化酶(G/Go)处理的C57BL/6乳小鼠心肌细胞(NMVCs)和心肌成纤维细胞(mCFs)中的表达;利用CCK8细胞增殖实验、流式细胞术和纤维化相关蛋白(包括COL1A1、COL3A1和α-SMA)表达检测来了解miR-208b-3p对mCFs纤维化表型的影响;双萤光素酶报告基因实验检测miR-208b-3p与潜在靶基因Mtf2和Pgrmc1 3′端非翻译区(3′-UTR)的结合作用;利用RT-qPCR和Western blot法分别检测miR-208b-3p转染的mCFs中Mtf2和Pgrmc1表达;通过小干扰RNA(siRNA)抑制Mtf2和Pgrmc1表达,并检测对mCFs中纤维化相关蛋白表达的作用。【结果】miRNA表达谱和RTqPCR结果证实miR-208b-3p在糖尿病db/db小鼠心肌中表达显著升高。miR-208b-3p前体与宿主基因Myh7在db/db小鼠心肌中表达显著升高,miR-208b-3p与Myh7 mRNA在乳小鼠来源的mCFs和NMVCs中均有表达,但在NMVCs中水平更高。miR-208b-3p在AngⅡ和G/Go处理后的mCFs和NMVCs中表达均明显升高。miR-208b-3p可显著增加mCFs中纤维化相关蛋白COL1A1、COL3A1和α-SMA表达,但不影响mCFs的增殖能力和细胞周期分布特征。双萤光素酶报告基因实验显示miR-208b-3p与Mtf2和Pgrmc1 3′-UTR有结合作用。miR-208b-3p可在转录后水平抑制mCFs中Mtf2和Pgrmc1表达。miR-208b-3p、Mtf2 siRNA和Pgrmc1 siRNA均能一致性地促进mCFs中纤维化相关蛋白表达。【结论】miR-208b-3p通过靶向Mtf2和Pgrmc1基因发挥促进mCFs中纤维化相关基因表达的作用。

【Objective】To investigate the effect of myosin heavy chain 7 gene-derived miRNA-208b-3p on the fibrot⁃ic phenotype of cardiac fibroblasts.【Methods】miRNA chip array was performed to detect the dysregulated miRNAs in the myocardium of diabetic db/db mice and db/m control mice.Neonatal mouse ventricular cardiomyocytes(NMVCs)and car⁃diac fibroblasts(CFs)were isolated from C57BL/6 mice and cultured.Real-time quantitative PCR(RT-qPCR)was con⁃ducted to determine the expression of miR-208b-3p in mouse CFs and NMVCs subjected to angiotensinⅡ(AngⅡ)and high glucose plus glucose oxidase(G/Go)treatment,respectively.Cell counting kit 8(CCk8)assay,flow cytometry and determination of fibrosis-related protein,including COL1A1,COL3A1andα-SMA,were performed in mCFs transfected with miR-208b-3p.Dual luciferase reporter assay was performed to confirm the interaction between miR-208b-3p and the 3′-UTR of metal response element binding transcription factor 2(Mtf2)and progesterone receptor membrane component 1(Pgrmc1),respectively.The expressions of Mtf2 and Pgrmc1 at the mRNA and protein levels in mCFs after miR-208b-3p mimic transfection were determined using RT-qPCR and Western blot assay,respectively.The small interfering RNA(siRNA)was used to inhibit Mtf2 and Pgrmc1 expression in mCFs,and the effects of Mtf2 siRNA,Pgrmc1 siRNA and miR-208b-3p on fibrosis-related protein expression in mCFs were investigated.【Results】Results of miRNA chip array and RT-qPCR assay showed that miR-208b-3p was up-regulated in the myocardium of the diabetic db/db mice.miR-208b precursor and the host gene of Myh7 were consistently increased in db/db mice.miR-208b-3p and Myh7 mRNA were expressed in mCFs and NMVCs,but the levels of miR-208b-3p and Myh7 mRNA in NMVCs were much higher than those in mCFs.miR-208b-3p was up-regulated in mCFs and NMVCs subjected to AngⅡand G/Go treatment,respective⁃ly.miR-208b-3p could significantly enhance fibrosis-related protein,including COL1A1,COL3A1 andα-SMA,in mCFs,without affecting the proliferation activity and cell cycle distribution of mCFs.Dual luciferase reporter assay re⁃vealed the interactions of miR-208b-3p with the 3′-UTR of Mtf2 and Pgrmc1.The results of RT-qPCR and Western blot⁃ting confirmed that miR-208b-3p inhibited Mtf2 and Pgrmc1 expression at the post-transcriptional level.Transfection with miR-208b-3p mimic,Mtf2 siRNA and Pgrmc1 siRNA could consistently enhance the fibrosis-related protein expres⁃sion in the cardiac fibroblasts.【Conclusions】miR-208b-3p enhances fibrosis-related gene expression by targeting Mtf2 and Pgrmc1in mCFs.