摘要
目的利用蛋白质组学技术考察知母皂苷AⅢ抑制小鼠脑胶质瘤细胞GL261(简称“GL261细胞”)增殖的分子机制。方法①将GL261细胞分为空白对照组(知母皂苷AⅢ0μmol/L)和不同剂量知母皂苷AⅢ给药组(1、2、4、6、8、10μmol/L),同时设置溶媒对照组[0.05%二甲基亚砜(DMSO)]和阳性药物组(紫杉醇,1μmol/L),按照分组分别将药液加入含10%胎牛血清(FBS)的达尔伯克改良伊格尔(DMEM)培养基中,混匀依次加入96孔板中,孵育72 h,采用四甲基偶氮唑盐微量酶反应比色法(MTT法)检测各组细胞存活率。②将GL261细胞分为空白对照组、知母皂苷AⅢ给药组(3.5、7μmol/L),按照分组分别将药液加入含10%FBS的DMEM中,混匀后依次加入96孔板中,孵育24 h,采用平板克隆法测定细胞的克隆形成,流式细胞术检测细胞凋亡情况;采用蛋白质组学技术分析潜在的靶标蛋白和相关的信号通路,并使用蛋白免疫印迹法对关键蛋白进行验证。结果与空白对照组比较,知母皂苷AⅢ呈剂量依赖性显著抑制GL261细胞活力和克隆形成,同时诱导GL261细胞凋亡(P<0.05)。蛋白质组学结果显示,知母皂苷AⅢ处理GL261细胞后,共有16个差异蛋白,其中5个蛋白上调,11个蛋白下调。基因本体(GO)数据库分析显示,主要生物进程、细胞组成和分子功能集中在增殖调节、生长负调控、细胞器、蛋白质结合、蛋白质同源二聚体活性等。京都基因与基因组百科全书(KEGG)数据库分析显示,主要通路富集在黏蛋白型O-聚糖生物合成、非小细胞肺癌、细菌侵袭表皮细胞、破骨细胞分化、FoxO信号通路、Wnt信号通路、紧密连接、细胞黏附分子、癌症中的蛋白聚糖、Ras信号通路。蛋白质免疫印迹法的结果显示,与空白对照组比较,知母皂苷AⅢ呈剂量依赖性抑制GL261细胞中CD276蛋白及Wnt信号通路中关键蛋白β-连环蛋白(β-catenin)、原癌基因蛋白(c-Myc)、细胞周期蛋白D1(CyclinD1)的表达(P<0.05)。结论知母皂苷AⅢ能抑制GL261细胞体外增殖、克隆形成,并诱导GL261细胞凋亡,其机制可能是通过抑制CD276蛋白表达,调控Wnt信号通路,从而发挥其抑制作用。
Objective The molecular mechanism of timosaponin AⅢinhibiting the proliferation on murine glioblastoma GL261(GL261 cell)was investiated by proteomics.Methods①GL261 cells were divided into blank control group(timosaponin AⅢ0μmol/L)and different dose groups of timosaponin AⅢ(1,2,4,6,8,10μmol/L).And solvent control group[0.05%dimethyl sulfoxide(DMSO)]and positive drug group[taxol(TAX),1μmol/L]were set up.According to the grouping,the solution was added to DMEM containing 10%fetal bovine serum(FBS),and then mixed into 96-well plate.After incubation for 72 h,the cell survival rate of each group was detected by MTT method.②GL261 cells were divided into blank control group and timosaponin AⅢgroup(3.5,7μmol/L).According to the grouping,the solution was added to DMEM containing 10%FBS,mixed and then added to 96-well plate for 24 h.The colony formation of the cells was determined by plate cloning,apoptosis detected by flow cytometry,and the potential target proteins and related signal pathways analyzed by proteomics.Western blot was used to verify the key proteins.Results Compared with the blank control group,timosaponin AⅢsignificantly inhibited the viability and colony formation of GL261 cells and induced apoptosis of GL261 cells in a dose-dependent manner.Proteomic results showed that there were 16 differential proteins in GL261 cells treated with timosaponin AⅢ,of which 5 proteins were up-regulated and 11 proteins were down-regulated.Gene ontology(GO)analysis showed that the main biological processes,cell composition and molecular functions focused on proliferation regulation,negative growth regulation,organelles,protein binding,protein homodimer activity and so on.Kyoto Encyclopedia of Gene and Genome(KEGG)analysis showed that the main pathways were concentrated in mucin O-glycan biosynthesis,non-small cell lung cancer,bacterial invasion of epidermal cells,osteoclast differentiation,FoxO signal pathway,Wnt signal pathway,tight junction,cell adhesion molecules,proteoglycan in cancer and Ras signal pathway.The results of Western blot showed that compared with the blank control group,timosaponin AⅢinhibited the expressions of CD276 protein and the key proteins in Wnt signaling pathway,such asβ-catenin,proto-oncogene(c-Myc)and cyclin D1(CyclinD1)in GL261 cells in a dose-dependent manner(P<0.05).Conclusions Timosaponin AⅢcan inhibit the proliferation and colony formation of GL261 cells in vitro and induce apoptosis of GL261 cells.The mechanism may be that it inhibits the expression of CD276 protein and regulates the Wnt signal pathway.
作者
常青祺
陈龙
袁春露
廖雅芳
郭玉刚
孙辉
章丹丹
CHANG Qingqi;CHEN Long;YUAN Chunlu;LIAO Yafang;GUO Yugang;SUN Hui;ZHANG Dandan(Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Experiment Center for Science and Technology,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Emergency Department of Jiangwan Hospital,Hongkou District,Shanghai,Shanghai 200433,China;Department of Oncology,Shanghai Lung Hospital Affiliated to Tongji University,Shanghai 200433,China)
出处
《上海中医药杂志》
2022年第8期71-78,共8页
Shanghai Journal of Traditional Chinese Medicine
基金
国家自然科学基金项目(81773946,81573673,81001666)
