| 271 | 1 | 77 |
| 下载次数 | 被引频次 | 阅读次数 |
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-1 24 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<0.000 1),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<0.000 1);CCK-8实验结果显示，TGF-β1组中HSCs的增殖能力增强(P<0.000 1);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<0.000 1);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<0.000 1);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<0.000 1);免疫荧光实验结果显示，TGF-β1+si-YTHDF1组中Mito-Tracker Red染色与Fis1共定位信号减少(P<0.01);JC-1染色实验结果显示，TGF-β1+si-YTHDF1组中线粒体膜电位下降(P<0.05)。结论 YTHDF1通过正向调控Fis1介导的线粒体裂变，促进了HSCs的活化、增殖和迁移能力。提示YTHDF1可能是参与调控HSCs活化、增殖和迁移的关键基因。
Abstract：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 for 24 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, type Ⅰ collagen(Collagen Ⅰ) and α-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<0.000 1), as well as the fibrosis markers Collagen Ⅰ and the expression level of α-SMA increased(P<0.01; P<0.001, P<0.000 1); while adding CCK-8, the experimental results showed that the proliferation ability of HSCs in the TGF-β1 group was enhanced(P<0.000 1); 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<0.000 1); 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 increased in the TGF-β1 group(P<0.01). Compared with the TGF-β1+si-NC group, RT-qPCR and Western blot experimental results showed that the expression of YTHDF1 and Fis1 in the TGF-β1+si-YTHDF1 group was reduced(P<0.01; P<0.001), and fibrosis markers the levels of CollagenⅠ and α-SMA were reduced(P<0.01; P<0.001, P<0.01).CCK-8 experimental results showed that the proliferation ability of HSCs in the TGF-β1+si-YTHDF1 group was weakened(P<0.000 1); Transwell experiment results showed that the migration ability of HSCs in the TGF-β1+si-YTHDF1 group was weakened(P<0.001); cell scratch experiment results showed that the migration ability of HSCs in the TGF-β1+si-YTHDF1 group was weakened(P<0.000 1); immunofluorescence experiment results showed that the Mito-Tracker Red staining and Fis1 co-localization signal decreased in the TGF-β1+si-YTHDF1 group(P<0.01); JC-1 staining experiment results showed that mitochondrial membrane potential decreased in the TGF-β1+si-YTHDF1 group(P<0.05).Conclusion YTHDF1 promotes the activation, proliferation and migration capabilities of HSCs by positively regulating Fis1-mediated mitochondrial fission. This suggests that YTHDF1 may be a key gene involved in regulating the activation, proliferation and migration of HSCs.
[1] Roehlen N,Crouchet E,Baumert T F.Liver fibrosis:mechanistic concepts and therapeutic perspectives[J].Cells,2020,9(4):875.doi:10.3390/cells9040875.
[2] You H,Ma X,Efe C,et al.APASL clinical practice guidance:the diagnosis and management of patients with primary biliary cholangitis[J].Hepatol Int,2022,16(1):1-23.doi:10.1007/s12072-021-10276-6.
[3] Toh M R,Wong E Y T,Wong S H,et al.Global epidemiology and genetics of hepatocellular carcinoma[J].Gastroenterology,2023,164(5):766-82.doi:10.1053/j.gastro.2023.01.033.
[4] 张亚楠，王璐瑶，周静，等.MiR-296-3p对胆总管结扎诱导的大鼠肝纤维化的影响[J].安徽医科大学学报，2024,59(9):1583-90.doi:10.19405/j.cnki.issn1000-1492.2024.09.013.[4] Zhang Y N,Wang L Y,Zhou J,et al.Effect of miR-296-3p on hepatic fibrosis induced by bile duct ligation in rats[J].Acta Univ Med Anhui,2024,59(9):1583-90.doi:10.19405/j.cnki.issn1000-1492.2024.09.013.
[5] Zhu H,Shan Y,Ge K,et al.Specific overexpression of mitofusin-2 in hepatic stellate cells ameliorates liver fibrosis in mice model[J].Hum Gene Ther,2020,31(1-2):103-9.doi:10.1089/hum.2019.153.
[6] Wang Y,Lu M,Xiong L,et al.Drp1-mediated mitochondrial fission promotes renal fibroblast activation and fibrogenesis[J].Cell Death Dis,2020,11(1):29.doi:10.1038/s41419-019-2218-5.
[7] Tian L,Potus F,Wu D,et al.Increased Drp1-mediated mitochondrial fission promotes proliferation and collagen production by right ventricular fibroblasts in experimental pulmonary arterial hypertension[J].Front Physiol,2018,9:828.doi:10.3389/fphys.2018.00828.
[8] Quan Y,Park W,Jin J,et al.Sirtuin 3 activation by honokiol decreases unilateral ureteral obstruction-induced renal inflammation and fibrosis via regulation of mitochondrial dynamics and the renal NF-κB-TGF-β1/smad signaling pathway[J].Int J Mol Sci,2020,21(2):402.doi:10.3390/ijms21020402.
[9] Hasan P,Saotome M,Ikoma T,et al.Mitochondrial fission protein,dynamin-related protein 1,contributes to the promotion of hypertensive cardiac hypertrophy and fibrosis in Dahl-salt sensitive rats[J].J Mol Cell Cardiol,2018,121:103-6.doi:10.1016/j.yjmcc.2018.07.004.
[10] Zhou Y,Long D,Zhao Y,et al.Oxidative stress-mediated mitochondrial fission promotes hepatic stellate cell activation via stimulating oxidative phosphorylation[J].Cell Death Dis,2022,13(8):689.doi:10.1038/s41419-022-05088-x.
[11] Nolden K A,Harwig M C,Hill R B.Human Fis1 directly interacts with Drp1 in an evolutionarily conserved manner to promote mitochondrial fission[J].J Biol Chem,2023,299(12):105380.doi:10.1016/j.jbc.2023.105380.
[12] 张利芬，李彬彬，余宏宇.MicroRNA-484通过靶向肝星状细胞中Fis1调控肝纤维化进程[J].第二军医大学学报，2017,38(9):1146-51.doi:10.16781/j.0258-879x.2017.09.1146.[12] Zhang L F,Li B B,Yu H Y.MicroRNA-484 regulates liver fibrosis course through targeting Fis1 in hepatic stellate cells[J].Acad J Second Mil Med Univ,2017,38(9):1146-51.doi:10.16781/j.0258-879x.2017.09.1146.
[13] Sikorski V,Selberg S,Lalowski M,et al.The structure and function of YTHDF epitranscriptomic m6A readers[J].Trends Pharmacol Sci,2023,44(6):335-53.doi:10.1016/j.tips.2023.03.004.
[14] Chen Z,Zhong X,Xia M,et al.The roles and mechanisms of the m6A reader protein YTHDF1 in tumor biology and human diseases[J].Mol Ther Nucleic Acids,2021,26:1270-9.doi:10.1016/j.omtn.2021.10.023.
[15] Fan S,Chen W X,Lv X B,et al.MiR-483-5p determines mitochondrial fission and cisplatin sensitivity in tongue squamous cell carcinoma by targeting FIS1[J].Cancer Lett,2015,362(2):183-91.doi:10.1016/j.canlet.2015.03.045.
基本信息:
DOI：10.19405/j.cnki.issn1000-1492.2025.01.007
中图分类号:R575.2
引用信息:
[1]贾琳,孙峰,董琪琪等.YTHDF1调控Fis1对肝星状细胞活化、增殖及迁移能力的影响[J].安徽医科大学学报,2025,60(01):49-58.DOI:10.19405/j.cnki.issn1000-1492.2025.01.007.
基金信息:
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)~~