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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 B member 11 (DNAJB 11) gene silencing on proliferation, apoptosis and cell cycle in human hepatocellular carcinoma SMMC7721 cells. METHODS: DNAJB11-shRNA lentiviral vector pCDH-Puro / DNAJB11-shRNA carrying DNAJB 11 gene was constructed and a recombinant lentivirus was prepared. Infection of SMMC7721 cells with a highly infectious recombinant virus. CCK-8 method was used to detect the proliferation of SMMC7721 cells after infection. Real-time fluorescent quantitative PCR and Western blotting were used to detect the expression of DNAJB11, proliferating cell nuclear antigen (PCNA) Gene caspase-3 mRNA and its protein expression; FCM method was used to detect the effect of silencing DNAJB 11 gene expression on the cycle and apoptosis of SMMC7721 cells. Results: Recombinant lentiviral vector pCDH-Puro / DNAJB11-shRNA with DNAJB11-shRNA inserted was successfully constructed. The results of CCK-8 assay showed that the proliferation of SMMC7721 cells was inhibited after infected with DNAJB11-shRNA recombinant lentivirus (P <0.05). The results of real-time fluorescence quantitative PCR and Western blotting confirmed that DNAJB11-shRNA could effectively silence DNAJB11 mRNA and protein expression in SMMC7721 cells (P <0.05). After silencing DNAJB 11, the expression of mRNA and protein of caspase-3 and the expression of PCNA mRNA and protein were down-regulated <0.05). The result of FCM showed that silenced DNAJB 11 gene could arrest SMMC7721 cells in G1 phase of cell cycle and promote apoptosis, the differences were statistically significant (P <0.01). Conclusion: Silencing DNAJB 11 gene expression can inhibit the proliferation and promote the apoptosis of SMMC7721 cells, which may be related to down-regulating the expression of PCNA and up-regulating the expression of caspase-3 in SMMC7721 cells.