摘要
目的利用siRNA方法沉默HepG2/ADM细胞系中BC047440基因,观察其受抑制后对细胞多药耐药性的影响及其分子机制。方法建立BC047440蛋白在HepG2/ADM细胞系siRNA干扰模型,采用Western blot检测BC047440蛋白的表达;实验分3组:BC047440-shRNA组、control-shRNA组和HepG2/ADM组。CCK-8法分析阿霉素对细胞生长抑制率的影响,流式细胞仪检测细胞凋亡及细胞周期分布;Western blot检测BC047440沉默后NF-κB蛋白表达的变化;Real-time PCR检测Survivin、CCNL1基因mRNA水平的变化。结果成功建立BC047440蛋白siRNA干扰模型;NF-κB蛋白表达BC047440-shRNA组约为HepG2/ADM组67.69%,约为control-shRNA组67.39%;细胞毒性实验中24、48 h阿霉素对BC047440-shRNA组细胞毒性作用明显增强;流式细胞仪检测结果显示BC047440-shRNA组细胞凋亡明显增多,细胞周期分布多停留于G1/G0期,S期细胞明显减少。Real-time PCR检测BC047440-shRNA组Survivin基因mRNA表达约为con-trol-shRNA组37%,约为HepG2/ADM组42%;检测BC047440-shRNA组CCNL1基因mRNA表达约为control-shRNA组3.5倍,约为HepG2/ADM组2.4倍。结论沉默BC047440基因可通过NF-κB信号通路及其下游Survivin、CCNL1信号逆转HepG2/ADM细胞的多药耐药性,提示BC047440基因可能是形成肝癌多药耐药的重要分子之一。
Objective To observe the effect of RNA interference （RNAi） -silenced BC047440 gene on muhidrug resistance of HepG2/ADM cells and its molecular mechanism. Methods BC047440 protein siRNA interference model was established in HepG2/ADM cells. Expression of BC047440 protein was detected by Western blotting. Experiment was conducted in BC047440-shRNA group, control-shRNA group and HepG2/ ADM group. Effect of adriamycin on cell growth was analyzed with CCK-8 method. Cell cycle and apoptosis were assayed by flow cytometry. Expression of NF-KB protein, Survivin mRNA and CCNL1 mRNA was detected by Western blotting and RT-PCR, respectively, after BC047440 was silenced. Results The siRNA interfer- ence model was successfully established. The NF-KB protein was expressed about 71.5% in BC047440-shRNA group and HepG2/ADM group, and about 70.0% in control-shRNA group. The eytotoxieity of adriamycin at 24 and 48 h was significantly higher in BC047440-shRNA group. Flow eytometry showed that the number of apop- totic cells was significantly greater in BC047440-shRNA group, the cell cycle was maintained at the G~/G0 phase, and the number of HepG2/ADM cells was reduced at the S phase. RT-PCR showed that the Survivin mRNA was expressed about 37% in control-shRNA group and about 42% in HepG2/ADM group, while the ex- pression of CCNL1 mRNA was about 3.5-fold higher in BC047440-shRNA group than in control-shRNA group and about 2.4-fold higher than in HepG2/ADM group. Conclusion Silencing BC047440 gene can reverse the multidrug resistance of HepG2/ADM cells though the NF-KB, and downstream survivin and CCNL1 signaling pathways, which indicates that BC047440 gene may be one of the important molecules for the multidrug resist- ance of liver cancer.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2012年第8期705-709,共5页
Journal of Third Military Medical University
基金
国家自然科学青年基金(81001105)~~
