YTHDF1调控Fis1对肝星状细胞活化、增殖及迁移能力的影响  

YTHDF1 regulation of Fis1 on the activation and proliferation and migration ability of hepatic stellate cells

作　　者：贾琳 孙峰 董琪琪 杨晶晶[2] 周仁鹏[1,2] 胡伟 鲁超[1,3] Jia Lin;Sun Feng;Dong Qiqi;Yang Jingjing;Zhou Renpeng;Hu Wei;Lu Chao(School of Pharmacy,Anhui Medical University,Hefei 230032;Dept of Clinical Pharmacology,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601;Dept of Clinical Pharmacology,The First Affiliated Hospital,Anhui University of Science and Technology,Huainan 232007)

机构地区：[1]安徽医科大学药学院,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,合肥230601 [3]安徽理工大学第一附属医院药物临床试验研究中心,淮南232007

出　　处：《安徽医科大学学报》2025年第1期49-58,共10页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金资助项目(编号:81600477);安徽高校自然科学基金资助项目(编号: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(编号:2019GMFY07);安徽省重点研究与开发计划项目(编号:202204295107020035);安徽省自然科学基金面上项目(编号:2208085MH215);合肥综合性国家科学中心大健康研究院职业医学与健康联合研究中心项目(编号:OMH-2023-03)。

摘　　要：目的探究YTH结构域家族蛋白1(YTHDF1)调控线粒体分裂蛋白1(Fis1)介导的线粒体裂变对肝星状细胞(HSCs)活化、增殖和迁移能力的影响。方法应用5 ng/ml TGF-β1处理小鼠肝星状细胞株JS-124 h以诱导其活化和增殖,并使用YTHDF1-siRNA构建YTHDF1沉默模型。实验分为Control组、TGF-β1组、TGF-β1+si-NC组和TGF-β1+si-YTHDF1组。通过逆转录定量聚合酶链反应(RT-qPCR)和蛋白质印迹(Western blot)实验检测YTHDF1、Fis1及纤维化关键指标Ⅰ型胶原蛋白(CollagenⅠ)、α-平滑肌肌动蛋白(α-SMA)的表达变化;利用CCK-8实验检测细胞增殖能力;通过Transwell迁移实验和细胞划痕实验检测细胞迁移能力;使用免疫荧光染色实验检测YTHDF1对Fis1介导的线粒体裂变的影响;最后通过JC-1染色实验检测YTHDF1对线粒体膜电位的影响。结果与Control组相比,RT-qPCR和Western blot实验结果显示,TGF-β1组中YTHDF1和Fis1的表达增加(P<0.05,P<0.01;P<0.0001),以及纤维化标志物CollagenⅠ和α-SMA的表达水平增加(P<0.01;P<0.001,P<0.0001);CCK-8实验结果显示,TGF-β1组中HSCs的增殖能力增强(P<0.0001);Transwell实验结果显示,TGF-β1组中HSCs的迁移能力增强(P<0.01);细胞划痕实验结果显示,TGF-β1组中HSCs的迁移能力增强(P<0.0001);免疫荧光实验结果显示,TGF-β1组中Mito-Tracker Red染色与Fis1共定位信号增加(P<0.05);JC-1染色实验结果显示,TGF-β1组中线粒体膜电位升高(P<0.01)。与TGF-β1+si-NC组相比,RT-qPCR和Western blot实验结果显示,TGF-β1+si-YTHDF1组中YTHDF1和Fis1的表达降低(P<0.01;P<0.001),纤维化标志物CollagenⅠ和α-SMA的水平降低(P<0.01;P<0.001,P<0.01),CCK-8实验结果显示,TGF-β1+si-YTHDF1组中HSCs的增殖能力减弱(P<0.0001);Transwell实验结果显示,TGF-β1+si-YTHDF1组中HSCs的迁移能力减弱(P<0.001);细胞划痕实验结果显示,TGF-β1+si-YTHDF1组中HSCs的迁移能力减弱(P<0.0001);免疫荧光实验结果显示,TGF-β1+si-YTHDF1组中Mito-Tracker Red染Objective To explore the effect of YTH domain family protein 1(YTHDF1)on the activation,proliferation and migration of hepatic stellate cells(HSCs)by regulating mitochondrial fission mediated by mitochondrial fission protein 1(Fis1).Methods The mouse hepatic stellate cell line JS-1 was treated with 5 ng/ml TGF-β1 for24 h to induce its activation and proliferation,and YTHDF1-siRNA was used to construct a YTHDF1 silencing model.The experiment was divided into Control group,TGF-β1 group,TGF-β1+si-NC group and TGF-β1+si-YTHDF1 group.Expression changes of YTHDF1,Fis1 and key indicators of fibrosis,typeⅠcollagen(CollagenⅠ)andα-smooth muscle actin(α-SMA)were detected through reverse transcription quantitative polymerase chain reaction(RT-qPCR)and Western blot;CCK-8 was used to detect cell proliferation ability;Transwell migration assay and cell scratch assay were used to detect cell migration ability;immunofluorescence staining experiment was used to detect the effect of YTHDF1 on Fis1-mediated mitochondrial fission;finally,JC-1 staining was used to experimentally detect the effect of YTHDF1 on mitochondrial membrane potential.Results Compared with the Control group,RT-qPCR and Western blot experimental results showed that the expression of YTHDF1 and Fis1 increased in the TGF-β1 group(P<0.05,P<0.01;P<0.0001),as well as the fibrosis markers CollagenⅠand the expression level ofα-SMA increased(P<0.01;P<0.001,P<0.0001);while adding CCK-8,the experimental results showed that the proliferation ability of HSCs in the TGF-β1 group was enhanced(P<0.0001);Transwell experimental results showed that the migration ability of HSCs in the TGF-β1 group was enhanced(P<0.01);the cell scratch experiment results showed that the migration ability of HSCs in the TGF-β1 group was enhanced(P<0.0001);the immunofluorescence experiment results showed that the TGF-β1 group Mito-Tracker Red staining and Fis1 co-localization signal increased(P<0.05);JC-1 staining experiment results showed that the mitochondrial membrane potential

关 键 词：YTHDF1 Fis1 HSCS 活化 增殖 迁移 肝纤维化 

分 类 号：R966[医药卫生—药理学]