UbcH10基因沉默对肺癌NCI-H226细胞增殖及细胞周期的影响

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表达载体，脂质体法转染重组质粒至NCI-H226细胞。转染后48小时，RT-PCR，Western-blot检测细胞内UbcH10 mRNA及蛋白含量。使用有效siRNA序列（pshRNA2）转染细胞，转染后24、48小时CCK-8检测细胞活性。使用有效siRNA序列转染细胞，转染后48小时收集细胞，流式法检测细胞周期。结果：成功构建shRNA重组质粒并转染NCI-H226细胞。转染siRNA 48小时后，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序列的沉默效果最好，UbcH10基因剩余表达量为对照组的14%。使用有效序列转染NCI-H226细胞24、48小时，细胞增殖活性明显降低，与基因未干预组比较，差异显著（P＜0.05）。使用有效序列转染NCI-H226细胞48小时，细胞明显阻滞于G2期，与基因未干预组比较有显著差异（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。

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

Objective: To silence UbcH10 gene in human lung squamous carcinoma cell line NCI-H226 using liposome transfection, and observe the changes in cell proliferative activity after gene silencing and the effect on cell cycle. Methods: Three siRNA sequences targeting different sites of UbcH10 CDS were designed, and the shRNA expression vectors were constructed. The recombinant plasmids were transfected into NCI-H226 cells by lipofectmaine2000; 48 hours after transfection, UbcH10 mRNA and protein levels were detected by RT-PCR and western blotting. NCI-H226 cells were transfected with the effective siRNA vector (pshRNA2), the cell viability was measured using CCK-8 24 or 48 hours after transfection and the cell cycle was detected by flow cytometry 48 hours after transfection. Results: the recombinant shRNA plasmids were successfully constructed and transfected into NCI-H226 cells. 48 hours after transfection, UbcH10 gene mRNA and protein in transfected NCI-H226 cells were decreased significantly, and the inhibitory effect of No. 2 siRNA sequence was the highest, reducing UbcH10 mRNA and protein by 86% and 82%. 24 and 48 hours after the transfection of NCI-H226 cells with the effective vector, the cell proliferative activity was decreased significantly (P<0.05), compared to the control group. 48 hours after the transfection, NCI-H226 cells were blocked in G2 phase, significantly different (P <0.05) to the control group. Conclusion: The silencing of UbcH10 gene can significantly inhibit the proliferative activity of NCI-H226 cells and block the cells in G2 phase.