DNMT3A调控Drp1对肝星状细胞活化增殖和迁移能力的影响  

Effects of DNMT3A regulating Drp1 on proliferation and migration of activated hepatic stellate cells

作　　者：王娟[1,2] 孙峰[1,2] 杨晶晶 鲁超[1,3] WANG Juan;SUN Feng;YANG Jing-jing;LU Chao(School of Pharmacy,Anhui Medical University,Hefei 230032,China;Dept of Clinical Pharmacology,the Second Hospital of Anhui Medical University,Hefei 230601,China;The First Affiliated Hospital,Anhui University of Science&Technology,Huainan Anhui 232001,China)

机构地区：[1]安徽医科大学药学院,安徽合肥230032 [2]安徽医科大学第二附属医院药物临床试验研究中心,安徽合肥230601 [3]安徽理工大学第一附属医院,安徽淮南232001

出　　处：《中国药理学通报》2022年第10期1542-1547,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81600477);安徽省高校自然科学基金资助项目(No KJ2020A0181);安徽医科大学第二附属医院国家自然科学基金孵育计划(No 2019GMFY07)。

摘　　要：目的探讨DNA甲基转移酶3A(DNA methyltransferase 3A,DNMT3A)调控动力相关蛋白1(dynamin-related protein 1,Drp1)介导的线粒体裂变对肝星状细胞(hepatic stellate cells,HSCs)活化增殖和迁移能力的影响。方法应用5μg·L^(-1)TGF-β1作用24 h诱导HSC-T6细胞活化,DNMT3A慢病毒感染构建DNMT3A沉默模型;实验分为Control组、TGF-β1组、TGF-β1+LV5-NC组和TGF-β1+LV5-DNMT3A组。分别用RT-qPCR和Western blot检测DNMT3A对相关mRNA和蛋白表达的影响;用CCK-8检测细胞活化增殖的情况;分别用细胞划痕、Transwell迁移实验观察DNMT3A对HSCs细胞迁移能力的影响。结果慢病毒感染成功构建DNMT3A沉默模型。与Control组比较,TGF-β1刺激后DNMT3A水平明显升高,纤维化标志物CollagenⅠ和α-SMA的mRNA和蛋白水平明显升高,线粒体裂变标志物Drp1的mRNA和蛋白水平明显升高,同时HSCs细胞的增殖和迁移能力明显增加;而与NC组比较,DNMT3A沉默组DNMT3A水平明显降低,CollagenⅠ、α-SMA和Drp1表达明显被抑制,HSCs细胞的增殖和迁移能力也明显被抑制。结论沉默DNMT3A可抑制Drp1的水平,同时抑制了HSCs细胞的增殖和迁移能力。提示DNMT3A介导低水平的DNA甲基化修饰可能通过抑制Drp1的水平来抑制线粒体裂变的发生,进而抑制HSCs活化,影响肝纤维化疾病的发生发展。Aim To investigate the effects of DNMT3A regulating Drp1 mediated mitochondrial fission on the proliferation and migration of active hepatic stellate cells.Methods HSC-T6 cells were activated by 5μg·L^(-1)TGF-β1 for 24 h,and DNMT3A lentivirus infection model was established to silence DNMT3A.The experiment was divided into control group,TGF-β1 group,TGF-β1+LV5-NC group and TGF-β1+LV5-DNMT3A group.The effects of DNMT3A on related mRNA and protein expression were detected by RT-qPCR and Western blot.The cell proliferation was detected by CCK-8.The effect of DNMT3A on the migration ability of HSCs cells was observed by Wound healing assay and Transwell migration assay.Results Lentivirus infection successfully constructed a DNMT3A silencing model.Compared with the control group,the level of DNMT3A significantly increased,the mRNA and protein levels of the fibrosis markers collagenⅠandα-SMA in the TGF-β1 group significantly increased,and the mRNA and protein levels of the mitochondrial fission marker Drp1 significantly increased;At the same time,the proliferation and migration ability of HSCs cells was significantly improved.Compared with the NC group,the DNMT3A level of the DNMT3A silenced group was significantly reduced,the expressions of collagen I,α-SMA and Drp1 were significantly inhibited,and the proliferation and migration capabilities of HSCs were also significantly inhibited.Conclusions Silencing DNMT3A inhibits the level of Drp1 and inhibits the proliferation and migration of HSCs at the same time.It is suggested suggest that DNMT3A-mediated low level DNA methylation modification may inhibit the occurrence of mitochondrial fission by inhibiting the level of Drp1,thereby inhibiting the activation of HSCs and affecting the occurrence and development of liver fibrosis.

关 键 词：肝纤维化 线粒体裂变 HSCS DNMT3A Drp1 增殖 迁移 

分 类 号：R-332[医药卫生] R329.24R329.28R342.3R575.2