摘要
目的:研究黄芩素介导黏着斑激酶(FAK)调控磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路影响胃癌HGC-27细胞增殖和迁移的作用和可能的分子机制。方法:使用黄芩素(0、5、15、25、50μmol·L^(-1))处理胃黏膜上皮GES-1细胞、胃癌HGC-27细胞48 h,噻唑蓝(MTT)比色法检测细胞增殖活力;蛋白免疫印迹法(Western blot)检测不同浓度黄芩素干预对FAK、上皮细胞-间质转化(EMT)及PI3K信号通路相关蛋白表达情况。利用慢病毒转染技术,构建稳定过表达FAK的胃癌HGC-27细胞,Western blot及绿色荧光蛋白(GFP)荧光检测FAK转染情况。细胞分为空载体组、黄芩素组、FAK过表达组和黄芩素处理FAK过表达组;MTT比色法检测细胞增殖活力;平板克隆形成实验观察克隆形成能力;Transwell迁移实验检测细胞迁移能力;Western blot检测EMT及PI3K信号通路相关蛋白表达情况。结果:与空白组比较,黄芩素(15、25、50μmol·L^(-1))组可明显抑制胃癌HGC-27细胞的增殖(P<0.05,P<0.01),并呈剂量依赖性,而对胃黏膜上皮GES-1细胞则未见明显抑制作用,黄芩素(5,15,25μmol·L^(-1))还可下调磷酸化(p)-FAK蛋白表达水平(P<0.05,P<0.01)。慢病毒转染HGC-27细胞后,与空载体组比较,FAK过表达组中FAK蛋白的相对表达量明显升高,GFP荧光拍照显示空载体组和FAK过表达组中HGC-27细胞均带有绿色荧光。MTT比色法、平板克隆形成实验及Transwell迁移实验检测结果显示,与空载体组比较,黄芩素组对HGC-27细胞的增殖、克隆形成和迁移具有抑制作用,FAK过表达组促进胃癌细胞的增殖、克隆形成和迁移能力。与FAK过表达组比较,使用黄芩素处理FAK过表达组则会抑制FAK增强的细胞增殖和迁移能力(P<0.05)。与空白组比较,黄芩素(15、25μmol·L^(-1))可明显上调胃癌HGC-27细胞E-钙黏蛋白(E-cadherin)蛋白表达,下调波形蛋白(Vimentin)、Snail、p-PI3K和p-Akt蛋白表达(P<0.05,P<0.01);与空载体组比较,FAK�
Objective:To study the possible molecular mechanism of baicalein(BAI)-mediated focal adhesion kinase(FAK)in the regulation of phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway to inhibit the proliferation and migration of gastric cancer HGC-27 cells.Method:The gastric epithelial GES-1 cells and gastric cancer HGC-27 cells were respectively treated with BAI(0,5,15,25,and 50μmol·L^(−1))for 48 h,and then methyl thiazolyl tetrazolium(MTT)assay was adopted to detect effect of BAI on cell proliferation.Western blot(WB)was employed to detect the expression of FAK and the proteins related to epithelial-mesenchymal transition(EMT)and PI3K signaling pathway after intervention with different concentrations of BAI.The HGC-27 cells stably overexpressing FAK were constructed with lentivirus-mediated transfection technique,and the transfection of FAK was detected through WB and green fluorescent protein(GFP).The cells were divided into empty vector(NC)group,BAI group,FAK overexpression group,and BAI-treated FAK overexpression group,and cell proliferation activity was detected by MTT assay.The colony formation and cell migration were observed via colony formation assay and Transwell migration assay,respectively.The expression of proteins involved in EMT and PI3K signaling pathways were detected by Western blot.Result:Compared with the NC group,BAI(15,25 and 50μmol·L^(−1))inhibited the proliferation of HGC-27 cells in a dose-dependent manner(P<0.05,P<0.01)while did not affect that of GES-1 cells.BAI(5,15 and 25μmol·L^(−1))down-regulated the expression level of p-FAK(P<0.05,P<0.01).Compared with NC group,FAK overexpression group showed up-regulated expression level of FAK in HGC-27 cells.The HGC-27 cells in both NC group and FAK overexpression group had green fluorescence.Compared with NC group,BAI inhibited the growth,colony formation,and migration,while FAK overexpression promoted those of HGC-27 cells.The treatment of FAK overexpression group with BAI inhibited the enhancement of cell prolife
作者
乔丹
张晟郡
王时玉
姚光媛
蔡英兰
陈丽艳
朴英实
QIAO Dan;ZHANG Sheng-jun;WANG Shi-yu;YAO Guang-yuan;CAI Ying-lan;CHEN Li-yan;PIAO Ying-shi(Cancer Research Center of Yanbian University,Key Laboratory of Pathobiology(Yanbian University),State Ethnic Affairs Commission,Research and Innovation Group of Yanbian University,Yanji 133002,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第7期73-80,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81860651)
吉林省大学生创新创业计划训练项目(202110184010)。
