论文部分内容阅读
目的通过脂质体转染siRNA方法沉默人肺鳞癌细胞NCI-H226中UbcH10基因,观察基因沉默后细胞增殖活性的变化及其对细胞周期的影响。方法设计3条针对UbcH10基因CDS区不同位点的siRNA序列并构建shRNA重组质粒,脂质体法转染重组质粒至NCI-H226细胞。转染后48 h,RT-PCR和蛋白质免疫印迹法检测细胞内UbcH10 mRNA及蛋白含量。使用有效siRNA序列转染细胞,转染后24、48 h CCK-8检测细胞活性。使用有效siRNA序列转染细胞,转染后48 h收集细胞,流式细胞术检测细胞周期。结果成功构建shRNA重组质粒并转染NCI-H226细胞。转染siRNA 48 h后,3组NCI-H226细胞中UbcH10基因的mRNA及蛋白含量均明显下降;其中2号siRNA序列(pshRNA2)的沉默效果最好,UbcH10基因剩余表达量为对照组的14%。使用pshRNA2转染NCI-H226细胞24、48 h,细胞增殖活性降低,与基因未干预组比较差异有统计学意义(P<0.05)。使用pshRNA2转染NCI-H226细胞48 h,细胞明显阻滞于G2期,与基因未干预组比较差异有统计学意义(P<0.05)。结论 UbcH10基因沉默可显著抑制NCI-H226细胞增殖活性,并使肺癌细胞阻滞于细胞G2期。
OBJECTIVE: To silence UbcH10 gene in human lung squamous cell carcinoma cell line NCI-H226 by siRNA transfection and observe the changes of cell proliferation and its effect on cell cycle after gene silencing. Methods Three siRNA sequences targeting different sites of CDS region of UbcH10 gene were designed and shRNA recombinant plasmids were constructed. The recombinant plasmids were transfected into NCI-H226 cells by lipofectamine. Forty-eight hours after transfection, the intracellular UbcH10 mRNA and protein levels were detected by RT-PCR and Western blotting. Cells were transfected with potent siRNA sequences and CCK-8 assayed for cell viability at 24 and 48 h post transfection. Cells were transfected with potent siRNA sequences and harvested 48 h after transfection. Cell cycle was assessed by flow cytometry. Results shRNA recombinant plasmid was successfully constructed and transfected into NCI-H226 cells. After transfection with siRNA for 48 h, the mRNA and protein levels of UbcH10 in three NCI-H226 cells were significantly decreased. Among them, the silencing effect of pshRNA2 was the best, and the remaining UbcH10 expression was 14% . The NCI-H226 cells were transfected with pshRNA2 for 24 and 48 h, the cell proliferation activity was decreased, and the difference was statistically significant (P <0.05) compared with the non-intervention group. NCI-H226 cells were transfected with pshRNA2 for 48 h, and the cells were arrested in G2 phase. The difference was statistically significant (P <0.05) compared with non-intervention group. Conclusion Silencing of UbcH10 can significantly inhibit the proliferation of NCI-H226 cells and arrest the proliferation of lung cancer cells in G2 phase.