毛花苷C诱导肝癌Huh-7细胞自噬并促进Beclin1与VPS34和VPS15的相互结合  

Lanatoside C induces autophagy in Huh-7 cells and promotes the combination of Beclin1 with VPS34 and VPS15

作　　者：王梦玲 吴丹[1] 谭盛强[2] 谭雄 郭昌淇 李鸿文[1] 谭宁[1] WANG Mengling;WU Dan;TAN Shengqiang;TAN Xiong;GUO Changqi;LI Hongwen;TAN Ning(College of Basic Medical Sciences of Guilin Medical University,Guilin 541199,Guangxi Zhuang Autonomous Region,China;Department of Hepatopancreatobiliary Surgery,Liuzhou People's Hospital Affiliated to Guangxi Medical University,Liuzhou 545006,Guangxi Zhuang Autonomous Region,China;Shatang Town Central Health Clinic,Liubei District,Liuzhou 545006,Guangxi Zhuang Autonomous Region,China;College of Clinical Medicine of Guilin Medical University,Guilin 541199,Guangxi Zhuang Autonomous Region,China)

机构地区：[1]桂林医学院基础医学院,广西壮族自治区桂林541199 [2]广西医科大学附属柳州市人民医院肝胆胰外科,广西壮族自治区柳州545006 [3]广西柳州市柳北区沙塘镇中心卫生院,广西壮族自治区柳州545006 [4]桂林医学院临床医学院,广西壮族自治区桂林541199

出　　处：《肿瘤》2024年第4期334-345,共12页Tumor

基　　金：国家自然科学基金地区基金项目(82160699);广西自然科学基金面上项目(2022JJA140859);广西壮族自治区教育厅2022年度广西高校中青年教师科研基础能力提升项目(2022KY0479);广西壮族自治区教育厅2021年研究生教育创新计划项目(JGY2021138);桂林医学院广西基础医学协同创新研究生联合培养基地建设项目资助[桂学位(2020)7号];2020年柳州市科技计划(2020NBAB0813);大学生创新创业训练国家级项(202110601005,202210601017)

摘　　要：目的:探究毛花苷C对肝癌Huh-7细胞自噬的影响,及可能的作用机制。方法:用梯度浓度的毛花苷C处理Huh-7细胞24 h后,采用CCK-8法检测毛花苷C对Huh-7细胞的IC_(50)值,并进一步通过CCK-8法和细胞克隆形成实验分析毛花苷C梯度浓度干预在Huh-7细胞增殖及克隆形成中的作用;采用蛋白质印迹法检测Huh-7细胞中自噬标志物微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)-Ⅱ、LC3-Ⅰ、p62、Beclin1、液泡分选蛋白15(vacuolar protein sorting 15,VPS15)、VPS34和自噬相关蛋白3(autophagy-related protein 3,ATG3)蛋白的表达水平,并观察LC3-Ⅱ/LC3-Ⅰ比值的变化情况;通过激光共聚焦显微镜观察毛花苷C对Huh-7细胞自吞噬小体形成的影响,并采用人工智能图像分析技术确认毛花苷C对Huh-7细胞自吞噬小体形成的影响;最后通过免疫共沉淀实验探究毛花苷C对Beclin1与VPS34以及VPS15相互作用的影响。结果:24 h时毛花苷C对Huh-7细胞的IC_(50)值为(0.49±0.07)μg/m L。CCK-8法和克隆形成实验检测结果提示,毛花苷C对Huh-7细胞的生长具有明显的抑制作用,且呈一定的浓度依赖性(P均<0.01)。蛋白质印迹法检测结果表明,随着毛花苷C浓度的提高,LC3-Ⅱ/LC3-Ⅰ比值也随之明显提高(P<0.01),同时Beclin1蛋白的表达水平被上调(P<0.05),而p62蛋白的表达水平被明显下调(P<0.01)。激光共聚焦显微镜下的观察结果显示,毛花苷C能够明显诱导Huh-7细胞自吞噬小体形成(P<0.01)。人工智能图像分析结果确认了毛花苷C的这一效应(P均<0.01)。免疫共沉淀实验结果证实,毛花苷C能够促进Beclin1与VPS34、VPS15相互结合(P均<0.01)。结论:毛花苷C明显抑制肝癌细胞Huh-7的增殖并诱导其发生自噬,其作用机制可能与毛花苷C上调Beclin1蛋白的表达水平并促进Beclin1与VPS34、VPS15相互结合密切相关。Objective:To investigate the effect of lanatoside C on autophagy in hepatocellular carcinoma Huh-7 cells,and the possible mechanism of action.Methods:After Huh-7 cells were treated with gradient concentration of lanatoside C for 24 h,the IC_(50)value of lanatoside C on Huh-7 cells was detected by CCK-8 assay,and the role of intervention of gradient concentration of lanatoside C in the proliferation and clone formation of Huh-7 cells was analyzed by CCK-8 assay and clone formation assay.Western blotting was used to detect the expression levels of autophagy markers in Huh-7 cells,such as microtubule-associated protein light chain 3(LC3)-II,LC3-Ⅰ,p62,Beclin1,vacuolar protein sorting 34(VPS30),VPS15 and autophagy-related protein 3(ATG3)in Huh-7 cells,and the changes of LC3-II/LC3-Ⅰratio were analyzed.The effects of lanatoside C on the formation of autophagic vesicles in Huh-7 cells were observed by laser confocal microscopy,and the effects of lanatoside C on the formation of autophagic vesicles in Huh-7 cells were confirmed by artificial intelligence image analysis.Finally,the effects of lanatoside C on the interaction between Beclin1,VPS34 and VPS15 on the formation of autophagic vesicles in Huh-7 cells were detected by co-immunoprecipitation.Results:The IC_(50)value of lanatoside C on Huh-7 cells at 24 h was(0.49±0.07)μg/mL.The results of the CCK-8 assay and clone formation assay suggested that lanatoside C had a significant inhibitory effect on the growth of Huh-7 cells in a concentration-dependent manner(both P<0.01).The results of Western blotting assay showed that the LC3-II/LC3-Ⅰratio was significantly increased with the increase in the concentration of lanatoside C(P<0.01),while the expression level of Beclin1 protein was up-regulated(P<0.05),and the expression level of p62 protein was significantly down-regulated(P<0.01).The results of laser confocal microscopy showed that lanatoside C could significantly induce autophagic vesicle formation in Huh-7 cells(P<0.01).The results of artificial intelligenc

关 键 词：肝癌 毛花苷C 自噬 BECLIN1 LC3-Ⅱ 

分 类 号：R735.7[医药卫生—肿瘤]