LMP1基因沉默对EBV阳性上皮细胞凋亡和增殖影响

APOPTOSIS OF EBV POSITIVE GASTRIC EPITHELIUM CELLS INDUCED BY siRNA TARGETED LMP1 GENE IS ASSOCIATED WITH INHIBITION OF Bcl-2 GENE EXPRESSION

目的探讨化学合成小干涉RNA(siRNA)对Epstein-Barr病毒(EBV)潜伏膜蛋白1(LMP1)阳性胃上皮(GT38)细胞LMP1编码基因表达的抑制作用,以及对细胞增殖和凋亡的影响。方法化学合成靶向LMP1mRNA不同位点的3对siRNA,分别转染GT38细胞,选择干扰效果最佳的siRNA进行实验研究,行RT-PCR、Hochest 33258荧光染色及流式细胞术分析。结果RT-PCR结果显示,3对靶向LMP1的siRNA均能抑制GT38细胞LMP1的转录表达,以靶向LMP1 mRNA 649位点的siRNA作用效果最强。Hochest 33258荧光染色可见siRNA649作用后的GT38细胞发生凋亡。流式细胞分析显示,siRNA649作用24、72以及120 h后的细胞其细胞周期无明显改变。RT-PCR检测结果表明,转染siRNA649的靶细胞Bcl-2的表达水平降低。结论靶向LMP1的特异性siRNA可以有效抑制LMP1的转录表达,进而可能通过抑制Bcl-2的表达诱导靶细胞凋亡。GT38细胞系可作为研究LMP1生物活性及其在EBV相关肿瘤发生中所起作用的理想的靶细胞。

Objective To explore the relationship between specific silencing effect of siRNA that targets latent membrane protein 1 （LMP1） coding gene on the proliferation and apoptosis of EBV positive gastric epithelium cells. Methods The chemically synthetic siRNA targeting LMP1 was transfected into target cells,then RT-PCR, Hochest 33258 staining and flow cytometry were used to detect apoptosis and cell cycle of target cells,respectively. Results RT-PCR showed that siRNAs markedly inhibited the expression of LMP1 in target cells,and the effect of siRNA649 was most obvious. Apoptosis was observed in target cells transfected by siRNA649 with Hochest 33258 staining. A flow cytometry analysis showed no obvious discrepancy after 24, 72 and 120 h of transfection of siRNA649. RT-PCR showed that siRNA649 inhibited the expression of Bcl-2 in target cells. Conclusion Chemically synthetic siRNA targeting LMP1 gene could effectively silence the expression of LMP1. Then it may though suppressing the expression of Bcl-2 lead target cells apoptosis. This cell line can be used as a goocl target cell to explore LMP1 bioactivlty and its effect in oncogenesis of EBV correlated tumors.