摘要
目的探讨靶向抑制DNA甲基转移酶1(DNMT1)对转化生长因子β1(TGF-β1)诱导的心肌成纤维细胞(CFs)纤维化的影响及其机制。方法采用DNMT1 siRNA重组慢病毒转染CFs,qRT-PCR和Western blot检测转染后的CFs中DNMT1表达变化。实验分组为对照组、TGF-β1组、si-NC+TGF-β1组、si-DNMT1+TGF-β1组、si-DNMT1+TGF-β1+3-甲基腺嘌呤(3-MA)组,CCK-8法测定CFs增殖活性,Transwell法检测CFs迁移数目,免疫荧光染色检测α-平滑肌肌动蛋白(α-SMA)表达情况,Western blot检测CFs中胶原Ⅰ型蛋白(COLⅠ)、胶原Ⅲ型蛋白(COLⅢ)、纤维连接蛋白(FN)及微管相关蛋白轻链3(LC3)、Beclin1的蛋白表达。结果DNMT1 siRNA重组慢病毒转染的CFs中DNMT1 mRNA相对表达量和蛋白相对表达量均显著下调(P<0.05)。与TGF-β1组比较,si-DNMT1+TGF-β1组细胞增殖活性显著降低,迁移数目显著减少,α-SMA相对荧光强度显著降低,COLⅠ、COLⅢ、FN蛋白相对表达量显著下调,LC3-Ⅱ/LC3-Ⅰ蛋白比值、Beclin1蛋白相对表达量显著上调(均P<0.05);而在si-DNMT1+TGF-β1组中同时加入自噬抑制剂3-MA处理后,细胞增殖活性未发生显著变化(P>0.05),但迁移数目显著增加,α-SMA相对荧光强度显著升高,COLⅠ、COLⅢ、FN蛋白相对表达量显著上调,LC3-Ⅱ/LC3-Ⅰ蛋白比值、Beclin1蛋白相对表达量显著下调(均P<0.05)。结论靶向抑制DNMT1能够改善TGF-β1诱导的CFs异常增殖与迁移,抑制纤维化,该作用可能与其促进自噬有关。
Objective To investigate the effect of targeted inhibition of DNA methyltransferase 1(DNMT1)on transforming growth factor β1(TGF-β1)-induced cardiac fibroblast(CFs)fibrosis and its mechanism.Methods Recombinant lentivirus with DNMT1 siRNA was used to transfect CFs,qRT-PCR and Western blot were used to detect the expression of DNMT1 in CFs after transfection.The experiment was divided into control group,TGF-β1 group,si-NC+TGF-β1 group,si-DNMT1+TGF-β1 group,si-DNMT1+TGF-β1+3-methyladenine(3-MA)group,the proliferative activity of CFs was determined by CCK-8 method,the migration number of CFs was detected by Transwell method,the expression ofα-smooth muscle actin(α-SMA)was detected by immunofluorescence staining,the protein expression of collagen type I(COLⅠ),collagen type Ⅲ(COLⅢ),fibronectin(FN),microtubule associated protein light chain 3(LC3)and Beclin1 were detected by Western blot.Results The relative expression of DNMT1 mRNA and protein in CFs infected with DNMT1 siRNA recombinant lentivirus were significantly down-regulated(P<0.05).Compared with TGF-β1 group,cell proliferation activity in si-DNMT1+TGF-β1 group was significantly decreased(P<0.05),the number of cell migration was significantly decreased(P<0.05),and the relative fluorescence intensity ofα-SMA was significantly decreased(P<0.05),the relative protein expressions of COLⅠ,COLⅢ and FN were significantly down-regulated(P<0.05),while the ratio of LC3-Ⅱ/LC3-Ⅰ protein and the relative protein expression of Beclin1 were significantly up-regulated(P<0.05).However,after the addition of autophagy inhibitor 3-MA in si-DNMT1+TGF-β1 group,there was no significant change in cell proliferation activity(P>0.05),but the number of cell migration was significantly increased(P<0.05),the relative fluorescence intensity ofα-SMA was significantly increased(P<0.05),and the relative protein expressions of COLⅠ,COLⅢ and FN were significantly up-regulated(P<0.05),the ratio of LC3-Ⅱ/LC3-Ⅰprotein and the relative protein expression of Beclin1 were significantly decreased(P<0.05).Conclusion Targeted inhibition of DNMT1 can improve the abnormal proliferation and migration of CFs induced by TGF-β1 and inhibit fibrosis,which may be related to the promotion of autophagy.
作者
成娜
姬佳妮
单梓梅
CHENG Na;JI Jiani;SHAN Zimei(Comprehensive Health Internal Medicine of Healthcare Center,Xinjiang Uygur Autonomous Region People's Hospital,Urumqi 830001,China;Healthcare Center,Xinjiang Uygur Autonomous Region People's Hospital,Urumqi 830001,China)
出处
《西部医学》
2025年第3期343-349,共7页
Medical Journal of West China
基金
新疆维吾尔自治区自然科学基金面上项目(2021D01C176)。
