沉默DNAJB11基因对肝癌SMMC7721细胞增殖、周期与凋亡的影响  

Effects of DNAJB11 gene-silencing on proliferation,cell cycle and apoptosis of human hepatocellular carcinoma SMMC7721 cells

作　　者：项颖[1] 李启英[1] 石洋[1] 张曼[1] 黄德鸿[1] 南映瑜[1] 杨涛[1] 肖春燕[1] 王莉[1] 张文军[1] 龚奕[1] 曾建挺[2] 张艳林[2] 

机构地区：[1]重庆市肿瘤研究所血液肿瘤生物治疗科,重庆400030 [2]重庆市肿瘤研究所肝胆外科,重庆400030

出　　处：《肿瘤》2017年第5期433-440,共8页Tumor

基　　金：重庆市应用开发计划资助项目(编号:cstc2014yykfA110031);中国医师协会研究项目(编号:ZGYSXH20120701)~~

摘　　要：目的:研究沉默DNAJ同源B亚家族成员11(DNAJ homolog subfamily B member 11,DNAJB 11)基因表达对人肝细胞癌SMMC7721细胞增殖、周期与凋亡的影响。方法:构建携带有针对DNAJB 11基因的DNAJB11-shRNA重组慢病毒载体pCDH-Puro/DNAJB11-shRNA,并制备重组慢病毒。将具有高效感染力的重组病毒感染SMMC7721细胞。CCK-8法检测被感染后SMMC7721细胞的增殖情况;分别采用实时荧光定量PCR法与蛋白质印迹法检测被感染后,SMMC7721细胞中DNAJB11、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和促凋亡基因caspase-3 mRNA及其蛋白的表达;FCM法检测沉默DNAJB 11基因表达后对SMMC7721细胞的周期和凋亡的影响。结果:成功构建插入DNAJB11-shRNA的重组慢病毒载体pCDH-Puro/DNAJB11-shRNA。CCK-8法检测结果显示,感染携带有DNAJB11-shRNA重组慢病毒后,SMMC7721细胞的增殖被抑制(P<0.05);实时荧光定量PCR法与蛋白质印迹法检测结果证实,DNAJB11-shRNA能有效沉默SMMC7721细胞中DNAJB11 mRNA及其蛋白的表达(P值均<0.05);DNAJB 11基因沉默后,能明显上调caspase-3 mRNA及蛋白的表达水平,并下调PCNA mRNA及蛋白的表达水平(P值均<0.05)。FCM检测结果表明,沉默DNAJB 11基因的表达可将SMMC7721细胞阻滞于细胞周期的G1期,并能促进其凋亡,差异均有统计学意义(P值均<0.01)。结论:沉默DNAJB 11基因表达可抑制SMMC7721细胞的增殖并促进其凋亡,这可能与下调SMMC7721细胞中增殖相关蛋白PCNA的表达,上调凋亡相关蛋白caspase-3的表达有关。Objective： To investigate the effects of DNAJ homolog subfamily B member 11 （DNAJB11） gene-silencing on proliferation, cell cycle and apoptosis of human hepatocellular carcinoma cell line SiVIMC7721. Methods： The recombinant lentiviral vector pCDH-Puro/DNAJB1 specific shRNA targeting DNAJB11 gene was established. The 1-shRNA carrying the SMMC7721 cells were infected with high infective lentivirus pCDH-Puro/DNAJB11-shRNA. Then the proliferation of SMMC7721 cells was detected by CCK-8 method. The expression levels of DNAJB11, proliferating cell nuclear antigen （PCNA） and caspase-3 mRNAs and proteins in SMMC7721 cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The cell cycle distribution and the apoptosis rate of SMMC7721 cells were analyzed by FCM. Results： The pCDH-Puro/DNAJB11-shRNA was constructed successfully. The proliferation of SMMC7721 ceils was significantly inhibited after infection with pCDH-Puro/DNAJB11- shRNA （P 〈 0.05）. In SMMC7721 cells infected with pCDH-Puro/DNAJB11-shRNA, the expressions of DNAJB11 mRNA and protein were silenced effectively （both P 〈 0.05）. After DNAJB11 gene-silencing, the expressions of caspase-3 mRNA and protein in SMMC7721 cells were up-regulated （both P 〈 0.05）, while the expressions of PCNA mRNA and protein were down-regulated （both P 〈 0.05）. Furthermore, the cell cycle was arrested in G1 phase （P 〈 0.01）, and the apoptosis rate was significantly increased （P 〈 0.01）. Conclusion： The DNAJBll gene-silencing can effectively suppress the proliferation of SMMC7721 cells, and promote their apoptosis. These effects may be related to downregulation of PCNA expression and upregulation of caspase-3 expression in SMMC7721 cells.

关 键 词：癌 肝细胞 DNAJB11基因 RNA 小分子干扰 细胞增殖 细胞凋亡 

分 类 号：R735.7[医药卫生—肿瘤]