UbcH10基因沉默对肺癌NCI-H226细胞增殖及细胞周期的影响

Effect of UbcH10 gene silencing on cell proliferation and cell cycle of lung cancer cell line NCI-H226

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 use liposomal siRNA for silencing UbcH10 gene in human lung squamous carcinoma cell line NCI-H226 and to observe the changes of cell proliferation and cell cycle after silencing.Methods Three siRNA sequences targeting different sites of UbcH10 CDS were designed,and the shRNA recombinant plasmids were constructed.The recombinant plasmids were transfected into NCI-H226 cells via lipofectamine2000.UbcH10 mRNA and protein expression was examined by RT-PCR and Western blotting analysis 48 h after transfection,respectively.The viability of NCI-H226 cells was measured using CCK-8 at 24 and 48 h after transfection with the effective siRNA vector,and the cell cycle was detected by flow cytometry 48 h after transfection.Results The recombinant shRNA plasmids were successfully constructed and transfected into NCI-H226 cells. UbcH10 gene mRNA and protein were noticeably decreased in the three groups 48 h after transfection,with the inhibitory effect of No.2 siRNA sequence（pshRNA2） being the strongest one（86%）.The proliferative activity of NCI-H226 cells was significantly decreased 24 and 48 h after the transfection with the pshRNA2 compared with the control group（P〈0.05）.Compared with the control group,NCI-H226 cells were blocked in G2 phase 48 h after the transfection with pshRNA2（P〈0.05）.Conclusion UbcH10 gene silencing can significantly inhibit the proliferative activity of NCI-H226 cells and block the cells in G2 phase.