SET7对Ang Ⅱ介导的心肌成纤维细胞增殖和胶原合成的影响及其机制

Effects of SET7 on angiotensin Ⅱ-mediated proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms

目的:沉默含SET结构域赖氨酸甲基转移酶7(SET domain containing lysine methyltransferase 7,SET7)基因可改善缺血再灌注介导的心肌组织损伤,但是SET7对血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)介导的心肌成纤维细胞增殖及胶原合成的影响尚不明确。本研究旨在探讨SET7对心肌成纤维细胞增殖以及胶原合成的影响及其可能机制。方法:分离心肌成纤维细胞,采用免疫荧光法鉴定心肌成纤维细胞。将心肌成纤维细胞随机分为4组:对照组,正常培养细胞;Ang Ⅱ组,给予100 nmol/L Ang Ⅱ处理细胞24 h;siCtrl组,细胞转染siCtrl后给予100 nmol/L的Ang Ⅱ处理24 h;siSET7组,细胞转染siSET7后给予100 nmol/L的Ang Ⅱ处理24 h。采用CCK-8法和5-乙炔基-2’-脱氧尿苷(5-ethynyl-2’-deoxyuridine,EdU)法检测细胞增殖情况;real-time PCR检测SET7、collagen Ⅰ、collagen Ⅲ和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)mRNA的表达;蛋白质印迹法检测SET7、collagen Ⅰ、collagen Ⅲ、音猬因子(sonic hedgehog,Shh)、跨膜蛋白patched1(Ptch1)、胶质瘤相关癌基因1(glioma-associated oncogene homolog 1,Gli1)和α-SMA蛋白质的表达。结果:在荧光显微镜下可见vimentin染色呈阳性,心肌成纤维细胞状态良好。与对照组比较,Ang Ⅱ组SET7 mRNA和蛋白质表达水平均显著上调,细胞增殖率和EdU荧光强度显著增加,collagen Ⅰ、collagen Ⅲ和α-SMA mRNA和蛋白质表达水平显著上调(均P<0.05);与siCtrl组比较,siSET7组SET7 mRNA表达水平显著下调,细胞增殖率和EdU荧光强度显著下降,collagen Ⅰ、collagen Ⅲ和α-SMA mRNA和蛋白质表达水平显著下调(均P<0.05)。结论:沉默SET7基因可抑制Ang Ⅱ介导的心肌成纤维细胞增殖和胶原合成,Shh信号通路可能参与这一过程。

Objective:Silence of SET domain containing lysine methyltransferase 7(SET7)alleviates myocardial tissue injury caused by ischemia-reperfusion.But the effects of SET7 on angiotensinⅡ(AngⅡ)-induced myocardial fibroblast proliferation and the collagen synthesis are not clear.The purpose of this study was to explore the effect of SET7 on the proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms.Methods:Myocardial fibroblasts were isolated and identified by immunofluorescence.Myocardial fibroblasts were randomly divided into 4 groups:a control group(cells were normally cultured),an AngⅡgroup(cells were treated with 100 nmol/L AngⅡfor 24 h),a siCtrl group(cells were transfected with siRNAcontrol and were then treated with 100 nmol/L AngⅡfor 24 h),and a siSET7 group(cells were transfected with siRNA SET7 and were then treated with 100 nmol/L AngⅡfor 24 h).Cell counting kit-8(CCK-8)and 5-ethynyl-2’-deoxyuridine(EdU)assay were used to evaluate cell proliferation.Real-time PCR was used to detect the mRNA levels of SET7,collagenⅠ,collagenⅢ,andα-smooth muscle actin(α-SMA).Western blotting was used to detect the protein expression of SET7,collagen Ⅰ,collagenⅢ,α-SMA,sonic hedgehog(Shh),ptched1(Ptch1),and glioma-associated oncogene homolog 1(Gli1).Results:Fluorescence microscopy showed positive vimentin staining,and myocardial fibroblasts were in good condition.As compared to the control group,the mRNA and protein levels of SET7 in the AngⅡgroup were significantly upregulated;cell proliferation rate and EdU fluorescence intensity in the AngⅡgroup were significantly increased;the mRNA and protein levels of collagenⅠ,collagenⅢ,andα-SMA were significantly upregulated(all P<0.05).As compared to the siCtrl group,the mRNA and protein levels of SET7 in the siSET7 group were significantly downregulated;cell proliferation rate and EdU fluorescence intensity in the siSET7 group were significantly decreased;the mRNA and protein levels of collagen Ⅰ,collagenⅢ,andα-SMA in the siSET7 group were significantly downregulated(all P<0.05).Conclusion:Silence of SET7 gene inhibits AngⅡ-induced proliferation and collagen synthesis of myocardial fibroblasts.Shh signaling pathway may be involved in this process.