WZB117下调YAP影响胃癌细胞干性和糖酵解的机制  

Mechanism of the effect of WZB117 on stemness and glycolysis via downregulation of YAP in gastric cancer cells

作　　者：张晨嵩 潘成武[1] 王庆康 李靖[1] 汪虎 马家驰 Zhang Chensong;Pan Chengwu;Wang Qingkang(Dept of Surgical Oncology Surgery,General Ward, The First Affiliated Hospital ofBengbu Medical College, Bengbu 233010)

机构地区：[1]蚌埠医学院第一附属医院肿瘤外科综合病区,蚌埠233010

出　　处：《安徽医科大学学报》2021年第8期1241-1246,共6页Acta Universitatis Medicinalis Anhui

基　　金：安徽高校自然科学研究项目(编号:KJ2019A0396);蚌埠医学院自然科学重点项目(编号:BYKY2019099ZD);蚌埠医学院胃癌多学科诊疗创新团队项目(编号:BYKC201907);蚌埠医学院自然科学基金重点项目(编号:2020byzd083)。

摘　　要：目的探究糖酵解抑制剂WZB117通过下调Yes相关蛋白(YAP)影响病人来源的胃癌细胞干性和糖酵解的机制。方法胰蛋白酶消化临床获取的胃癌组织,培养制备胃癌细胞;细胞分为正常组、低糖对照组和WZB117处理组,qRT-PCR检测细胞干性相关基因(Nanog、oct-4、sox-2)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)和凋亡相关基因(Bcl-2、bax和Cyt-C)的表达;Western blot测定细胞中YAP和代谢相关蛋白己糖激酶2(HK2)、丙酮酸激酶M2亚型(PKM2)和活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)的表达;MTT测定细胞增殖活性;平板克隆实验测定细胞克隆形成能力;划痕试验观察细胞迁移能力;Transwell实验观察细胞的侵袭能力;试剂盒测定细胞中三磷酸腺苷(ATP)及培养液上清液中的乳酸含量。结果WZB117培养细胞后,YAP表达水平较正常组及低糖对照组显著降低(P<0.05);Nanog、BMil、c-Myc的表达以及细胞增殖活性、克隆形成率、侵袭能力、迁移能力降低(P<0.05);Cleaved Caspase-3水平及细胞凋亡率升高(P<0.05);Bcl-2表达随WZB117浓度增高而降低(P<0.05);Bax和Cyt-C表达随WZB117浓度增高而升高(P<0.05);ATP、上清液中乳酸含量以及HK2、PKM2蛋白水平降低(P<0.05)。结论抑制剂WZB117可通过下调胃癌细胞中YAP表达,干扰糖酵解关键酶HK2、PKM2的表达,抑制胃癌细胞糖酵解,降低细胞ATP水平。Objective To explore the mechanism by which the inhibitor WZB117 affects the stemness and glycolysis via the downregulation of Yes-related proteins in patient-derived gastric cancer cells.Methods Patient-derived gastric cancer cells were obtained via the trypsin digestion of clinical gastric cancer tissues and incubation.The cells were divided into normal group,low glucose control group and WZB117 treatment group.Afterwards,qRT-PCR was used to detect the expression levels of stemness-related genes(Nanog,oct-4 and sox-2),invasion-related genes matrix metalloproteinase-2(MMP-2)and matrix metalloproteinase-9(MMP-9),and apoptosis-related genes(Bcl-2,bax and Cyt-C).Western blot was applied to determine the expression levels of YAP,metabolism-related proteins hexokinase 2(HK2)and pyruvate kinase M2 subtype(PKM2)and activated cysteine-containing aspartate proteolytic enzyme 3(Cleaved Caspase-3).Thiazolyl blue(MTT),plate clone,scratch test,Transwell and kit were performed to measure the cell proliferation,clone formation,migration,invasion,and content of adenosine triphosphate(ATP)and lactic acid,respectively.Results After WZB117 cultured cells,the YAP expression level was significantly lower than that of the normal group and the low glucose control group(P<0.05),and that of stemness-related genes(Nanog,oct-4 and sox-2)and cell proliferation,clone formation,invasion,and migration were also lower(P<0.05).The levels of Cleaved Caspase-3 and apoptosis in group treated by WZB117 were higher than those in control groups(P<0.05),and the expression level of Bcl-2 decreased with the increase of WZB117 concentration(P<0.05),and the expression levels of Bax and CYT-C increased with the increase of WZB117 concentration(P<0.05).The content of ATP and lactic acid in supernatant and the protein expression levels of HK2 and PKM2 were lower than those in control groups.Conclusion WZB117 can down regulate the expression of Yap,HK2 and PKM2 in gastric cancer cells,inhibit glycolysis and reduce ATP level.

关 键 词：WZB117 胃癌 Yes相关蛋白 癌细胞干性 糖酵解 

分 类 号：R735.2[医药卫生—肿瘤]