摘要
目的:探究γ-氨基丁酸受体亚基α-3(Gamma-aminobutyric acid receptor subunit alpha-3,Gabra3)基因对膀胱癌T24细胞凋亡、迁移和侵袭的作用及其机制。方法:TCGA数据库分析Gabra3基因在膀胱癌中的表达以及转移和生存的相关性。建立Gabra3过表达和Gabra3突变的T24细胞株,根据转染质粒不同,分为空白T24细胞(Control)、空pDONR223载体转染T24细胞(NC)、野生型Gabra3过表达T24细胞株(wt-Gabra3)、突变型Gabra3 T24细胞株(mut-Gabra3)。在回补实验中,分为转染突变型Gabra3 T24组(mut-Gabra3)、转染空pDONR223载体mut-Gabra3组(mut-Gabra3-NC)、转染野生型Gabra3过表达组(mut-Gabra3+wt-Gabra3)。用TUNEL染色检测T24细胞凋亡,细胞划痕和Transwell分别检测T24细胞迁移和侵袭,Western blot检测AKT/mTOR通路相关分子生物表达水平。结果:Gabra3基因在膀胱癌中显著高表达(P<0.05),生存曲线证实远处转移存在的患者生存期明显较短(P<0.05)。成功构建Gabra3过表达和突变的T24细胞株。与Control组相比,wt-Gabra3组细胞凋亡能力显著降低,迁移、侵袭能力显著增强(P<0.05),而mut-Gabra3组细胞凋亡显著增加,侵袭能力显著减弱(P<0.05);wt-Gabra3组细胞p-AKT、p-mTOR、p-4E-BP1、p-S6K蛋白表达均显示上凋(P<0.05),而mut-Gabra3组细胞上述蛋白无差异。在回补实验中,过表达wt-Gabra3的mut-Gabra3突变T24细胞凋亡能力受到抑制(P<0.05),迁移和侵袭能力显著增强(P<0.05),并且该组细胞AKT/mTOR通路相关分子显著激活(P<0.05)。结论:外源性的未编辑的Gabra3可以促进膀胱癌T24细胞的迁移、侵袭能力,并且可能与激活AKT/mTOR途径通路密切相关。
Objective:To explore the role of Gamma-aminobutyric acid receptor subunit alpha-3(Gabra3)gene on apoptosis,migration and invasion of bladder cancer T24 cells and its mechanism.Methods:The TCGA database was used to analyze the expression of Gabra3 gene in bladder cancer and the correlation between metastasis and survival.T24 cell lines with Gabra3 overexpression and Gabra3 mutation were established.According to the transfection plasmid,T24 cells were divided into blank T24 cells(Control),T24 cells transfected with empty pDONR223 vector(NC),and T24 cells with wild type Gabra3 overexpression(wt-Gabra3),mutant Gabra3 T24 cell line(mut-Gabra3).In the complementation experiment,T24 cells were divided into mutant Gabra3(mut-Gabra3),mut-Gabra3 transfected with empty pDONR223 vector mut-Gabra3 group(mut-GABRA3-NC),and wild type Gabra3 overexpression group(mut-Gabra3+wt-Gabra3).The apoptosis of T24 cells was detected by TUNEL staining,the migration and invasion of T24 cells were detected by cell scratch and Transwell,respectively.The expression levels of AKT/mTOR pathway related molecules were detected by Western blot.Results:Gabra3 gene was significantly overexpressed in bladder cancer(P<0.05),and the survival curve confirmed that patients with distant metastasis had a significantly shorter survival(P<0.05).T24 cell lines with Gabra3 overexpression and mutation were successfully constructed.Compared with the Control group,the apoptosis ability of wt-Gabra3 group was significantly decreased,and the migration and invasion ability were significantly increased(P<0.05),while the mut-Gabra3 group showed significantly increased apoptosis and decreased invasion(P<0.05).The expression levels of p-AKT,p-mTOR,p-4E-BP1 and p-S6K proteins in wt-Gabra3 group were increased(P<0.05),while mut-Gabra3 group had no difference in the above proteins.In complementation assay,the mut-Gabra3 mutant T24 cells overexpressing wt-Gabra3 showed inhibited apoptosis(P<0.05),and the migration and invasion abilities were significantly enhanced(P<0.05).AK
作者
李明明
韩利忠
王亚楠
卢冠军
吕志勇
LI Mingming;HAN Lizhong;WANG Yanan;LU Guanjun;LYU Zhiyong(Department of Urology,General Hospital of Ningxia Medical University,Ningxia Yinchuan 750000,China)
出处
《现代肿瘤医学》
CAS
2024年第7期1208-1214,共7页
Journal of Modern Oncology
基金
宁夏自然科学基金(编号:2021AAC03325)。
