摘要
目的探究细胞命运决定子Numb调控胃癌细胞对多柔比星(ADR)敏感性的作用,并明确Numb介导胃癌细胞化疗敏感性的分子机制。方法将SGC-7901随机分为对照组、ADR组、pLVX-Numb组、pLVX-Numb+ADR组,经过对应的ADR处理与转染处理后,Hoechst 33258染色观察细胞凋亡形态,流式细胞术检测细胞凋亡率,Western blot法测定细胞内耐药蛋白(P-gp)、多聚ADP核糖聚合酶(PARP)、裂解的半胱氨酸天冬氨酸特异性蛋白酶3(Cleaved-caspase3)、Cleaved-caspase9的蛋白表达水平。再使用Numb过表达及其对照慢病毒感染SGC-7901细胞,qRT-PCR和Western blot检测细胞内ITGB1表达变化,免疫共沉淀实验分析细胞内源性Numb与ITGB1结合情况。结果与ADR组比较,pLVX-Numb+ADR组细胞内浓缩、碎裂的蓝色凋亡体增多,细胞凋亡率升高,且细胞内P-gp和PARP蛋白相对表达量下调、Cleaved-caspase3和Cleaved-caspase9蛋白相对表达量上调(P<0.05)。此外,SGC-7901细胞中内源性Numb与ITGB1能够形成复合物,pLVX-Numb组细胞中ITGB1蛋白相对表达量显著低于对照组(P<0.05),且ITGB1蛋白泛素链明显强于对照组。结论Numb能够促进胃癌细胞对多柔比星的敏感性,该作用机制可能与其调控ITGB1蛋白表达相关。
Objective To explore the role of the cell fate determinant Numb in regulating the sensitivity of gastric canceRcells to doxorubicin(ADR),and to clarify the moleculaRmechanism of Numb-induced chemosensitivity of gastric canceRcells.Methods SGC-7901 cells were randomly divided into control group,ADRgroup,pLVX-Numb group and pLVX-Numb+ADRgroup.AfteRcorresponding ADRtreatment and cell transfection,the morphology of apoptotic cells and the apoptosis rate were detected by Hoechst 33258 staining and flow cytometry,respectively.Protein levels of P-glycoprotein(P-gp),poly(ADP-ribose)polymerase(PARP),cleaved-cysteine aspartate specific protease-3(cleaved-caspase3)and cleaved-caspase9 in cells were determined by Western blot.SGC-7901 cells were then infected with Numb overexpression lentivirus and its control lentivirus,and the changes in the expression levels of integrin beta1(ITGB1)in cells were detected by quantitative reverse transcriptase polymerase chain reaction(qRT-PCR)and Western blot.The binding of endogenous Numb to ITGB1 was analyzed by co-immunoprecipitation assay.Results Compared with the ADRgroup,increased condensed and fragmented blue apoptotic bodies,higheRapoptosis rate,downregulated P-gp and PARP,and upregulated cleaved-caspase3 and cleaved-caspase9 were detected in the pLVX-Numb+ADRgroup(P<0.05).In SGC-7901 cells,endogenous Numb and ITGB1 could form complexes.The relative expression of ITGB1 in cells in pLVX-Numb group was significantly loweRthan that in the control group(P<0.05),and the ubiquitin chain of ITGB1 protein was significantly strongeRthan that in the control group.Conclusion Numb can promote the sensitivity of gastric canceRcells to ADRby regulating ITGB1.
作者
陈勇
何冬雷
周江浩
梁月祥
杨丞
CHEN Yong;HE Donglei;ZHOU Jianghao(Department of Gastrointestinal TumoRSurgery,the First Affiliated Hospital of Hainan Medical University,Hainan,Haikou 570102,China)
出处
《河北医药》
CAS
2023年第15期2256-2260,共5页
Hebei Medical Journal
基金
海南省卫生健康行业科研项目(编号:21A200052)。