YB-1基因沉默对肝癌细胞化学治疗敏感性的影响及可能机制

Effects and possible mechanism of YB-1 gene silencing on chemosensitivity of hepatocellular carcinoma cells

目的探讨沉默YB-1基因对肝癌细胞化学治疗敏感性的影响及可能机制.方法根据YB-1靶点序列设计YB-1基因siRNA干扰序列,与EcoRⅠ和BamHⅠ双酶切后的线性化pLKD-CMV-G&PR-U6慢病毒载体连接及转化形成重组慢病毒;参照此方法设计并构建阴性对照重组慢病毒,并将其转染肝癌细胞SMMC-7721,分别命名为YB-1-siRNA组和NC-siRNA组,设置空白对照组(未经任何处理的肝癌细胞SMMC-7721),分别加入含顺铂(1μg/ml)和多柔比星(1μg/ml)的现配培养基.采用实时荧光定量PCR检测各组药物处理前后YB-1 mRNA表达水平;采用MTT法检测药物处理前后各组细胞的增殖情况;采用流式细胞术检测药物处理前后各组细胞的凋亡情况,采用Western blot检测各组药物处理后PI3K、AKT和GSK-3β蛋白的表达.结果处理前后,YB-1-siRNA组、NC-siRNA组和空白对照组间YB-1 mRNA表达水平及细胞活力差异均有统计学意义(P<0.05),与处理前比较,处理后YB-1-siRNA组、NC-siRNA组和空白对照组YB-1 mRNA表达水平和细胞活力均显著下降(P<0.05).处理前后,YB-1-siRNA组、NC-siRNA组和空白对照组间细胞凋亡率的差异有统计学意义(P<0.05);与处理前比较,处理后各组细胞凋亡率均显著升高(P<0.05).经药物处理后,3组间PI3K、AKT和GSK-3β蛋白表达差异均有统计学意义(P<0.05).结论靶向敲除YB-1基因联合化学治疗可显著增强抑制肝癌细胞增殖及促进其凋亡的能力,可能与PI3K/AKT/GSK3β信号转导通路有关.

Objective To investigate the effects and possible mechanism of targeted silencing of YB-1 gene on chemosensitivity of hepatocellular carcinoma cells. Methods The siRNA interference sequence of YB-1 gene was designed according to the target sequence of YB-1. The recombinant lentivirus was formed by ligation and transformation of the siRNA interference sequence of YB-1 gene with the lentiviral vector pLKD-CMVG& PR-U6 after digestion with EcoRⅠ and BamHⅠ double enzymes. According to this method, the negative control recombinant lentivirus was designed and constructed and transfected into hepatocellular carcinoma cell line SMMC-7721, and named YB-1-siRNA group and NC-siRNA group, respectively. The blank control group was also set up (without any treatment of liver cancer cell SMMC-7721). Then the ready mixed medium with cisplatin (1 μg/ml) and doxorubicin (1 μg/ml) were added, respectively. Real-time fluorescent PCR was used to detect the expression of YB-1 mRNA in each group before and after drug treatment. MTT was used to detect the proliferation of cells in each group before and after drug treatment. Flow cytometry was used to detect the apoptosis of cells in each group before and after drug treatment. Western blot was used to detect the expression of PI3K, AKT and GSK-3β protein in each group after drug treatment. Results Before and after treatment, YB-1-siRNA group, NC-siRNA group and blank control group had significant differences in YB-1 expression level and cell viability (P < 0.05). Compared with pretreatment, YB-1 expression level and cell viability in YB-1-siRNA group, NC-siRNA group and blank control group decreased significantly after treatment (P < 0.05). Before and after treatment, the apoptotic rates of YB-1-siRNA group, NC-siRNA group and blank control group were significantly different (P < 0.05). Compared with before treatment, the apoptotic rates of YB-1-siRNA group, NC-siRNA group and blank control group increased significantly after treatment (P < 0.05). After drug treatment, there was significant differences in PI3K, AKT and GSK- 3β protein expression among three groups (P < 0.05). Conclusion YB-1 gene knocked out combined with chemotherapy can significantly enhance the ability of inhibiting proliferation and promoting the apoptosis of hepatocellular carcinoma cells, which may be related to PI3K/AKT/GSK3β signaling pathway.