下调Notch1基因促进人肝癌细胞系HepG2自噬  

Down-regulation of Notch1 gene promotes autophagy of human liver cancer cell line HepG2

作　　者：李长安[1] 刘春秀[1] 谢晓娟 孙爱平[2] LI Chang-an;LIU Chun-xiu;XIE Xiao-juan;SUN Ai-ping(Department of Infectious Diseases, the Third Affiliated Hospital of Xinxiang Medical College,Xinxiang 453000;Department of Immunology, Xinxiang Medical College, Xinxiang 453000, China)

机构地区：[1]新乡医学院第三附属医院感染科,河南新乡453000 [2]新乡医学院免疫学教研室,河南新乡453000

出　　处：《基础医学与临床》2021年第4期508-513,共6页Basic and Clinical Medicine

基　　金：国家科技重大专项课题(2013ZX10002005);河南省高等学校重点科研项目(20B310009)。

摘　　要：目的探讨Notch1基因对人肝癌细胞系HepG2的增殖、凋亡、自噬及Akt/mTOR信号通路的影响。方法选择于新乡医学院第三附属医院行肝癌手术治疗患者的肝癌组织32例,荧光定量PCR检测Notch1 mRNA的表达。构建Notch1 siRNA真核表达载体,经脂质体转染入HepG2细胞中,应用RT-qPCR及Western blot鉴定Notch1的干扰效果,MTT法检测细胞的增殖,流式细胞术检测细胞的凋亡率,Western blot检测凋亡相关蛋白BCL-2、BAX、cleared-caspase-3,自噬相关蛋白LC3B、Beelinl、ATG7、ATG5及Akt/mTOR信号通路相关蛋白Akt、p-Akt、p-mTOR、p-P70S6K的表达。结果Notch1基因在原发性肝细胞癌组织中的相对表达量(1.865±0.791)显著高于癌旁组织(0.709±0.414)(P<0.001)。Notch1 siRNA转染HepG2细胞后,Notch1 siRNA组中Notch1 mRNA及蛋白的表达均显著低于NC siRNA组和对照组(P<0.001)。Notch1 siRNA组24、48、72及96 h的细胞增殖率显著低于NC siRNA组和对照组(P<0.001)。转染48 h后,Notch1 shRNA组细胞的凋亡率及Bax水平显著高于NC siRNA和对照组,而Bcl-2、cleaved caspase-3的表达显著低于NC siRNA组和对照组(P<0.001)。Notch1 siRNA组细胞中自噬相关蛋白LC3B、Beelinl、ATG7、ATG5的表达量显著高于NC siRNA组和对照组(P<0.001)。而Notch1 siRNA组的p-Akt、p-mTOR、p-P70S6K表达量显著低于NC siRNA组和对照组(P<0.001)。结论下调Notch1基因的表达可显著抑制肝癌HepG2细胞的增殖,诱导细胞的凋亡,提高细胞的自噬水平,其机制可能与抑制Akt/mTOR信号通路有关。Objective To investigate the effect of Notch1 gene on the proliferation,apoptosis,autophagy and Akt/mTOR signaling pathway of HepG2 liver cancer cells.Methods Thirty-two cases of liver cancer tissues from patients undergoing liver cancer surgery in the Third Affiliated Hospital of Xinxiang Medical College were selected,and the expression of Notch1 mRNA was detected by fluorescence quantitative PCR.Notch1 siRNA eukaryotic expression vector was constructed,transfected into HepG2 cells by liposome,RT-qPCR and Western blot were applied to check the interference effect of Notch1,MTT method was used to detect cell proliferation and flow cyto-metry was used to detect cell apoptosis,Western blot was also applied in the detection of apoptosis-related proteins BCL-2,BAX,cleared-caspase-3,autophagy-related proteins LC3B,Beelinl,ATG7,ATG5 and Akt/mTOR signaling pathway related proteins Akt,p-Akt,p-mTOR,p-P70S6K.Results The relative expression of Notch1 gene in primary hepatocellular carcinoma tissue was significantly higher than that in adjacent tissues(1.865±0.791 vs 0.709±0.414)(P<0.001).After Notch1 siRNA was transfected into HepG2 cells,the expression of Notch1 mRNA and protein in Notch1 siRNA group was significantly lower than that in NC siRNA group and in control group(P<0.001).The cell proliferation rate of Notch1 siRNA group at 24,48,72 and 96 h was significantly lower than that of NC siRNA group and control group(P<0.001).Forty-eight hours after transfection,the apoptosis rate and Bax level of Notch1 shRNA group were significantly higher than that of NC siRNA and control group,and the expression of Bcl-2 and cleaved caspase-3 were significantly lower than that of NC siRNA group and control group(P<0.001).The expression of autophagy-related proteins LC3B,Beelinl,ATG7 and ATG5 in Notch1 siRNA group was significantly higher than that of NC siRNA group and control group(P<0.001).The expression level of p-Akt,p-mTOR,and p-P70S6K in Notch1 siRNA group was significantly lower than those in NC siRNA group and control grou

关 键 词：肝癌 NOTCH1基因 Akt/mTOR信号途径 自噬 

分 类 号：R34[医药卫生—基础医学]