目的 下调EZH2的表达,探讨EZH2在胃癌细胞株MKN1增殖、细胞周期中的作用,研究EZH2在胃癌发生与发展中的作用.方法 将EZH2的siRNA表达载体pSilencer 2.1-U6(+)通过Lipofect AUINE 2000转入人胃癌细胞MKN1中,G418选择培养,建立EZH2低表达的胃癌细胞系MKN1/Silence(+)；RT-PCR和Western blot检测EZH2在MKN1、MKN1/Silence(+)和MKN1/Silence(-)细胞表达,MTT法检测细胞增殖的活性,流式细胞观察其对MKN1细胞周期的影响.结果 构建成功稳定表达EZH2 siRNA细胞MKN1/Silence(+):EZH2在MKN1/Silenc(+)的mRNA和蛋白的表达明显下降,MKN1/Silence(+)细胞增殖明显抑制.在细胞周期试验中,MKN1/Silence(+)细胞G2/M期(36.08±1.34)比MKNI(16.59 ±0.71)、空载体MKNI/Silence(-)(18.35 ±0.35)显著增高(P＜0.01),Go/G1期细胞显著减少(P＜0.01),而S期细胞无明显变化,EZH2 siRNA作用下阻滞于G2/M期.结论 下调EZH2的表达显著性阻滞了MKN1细胞的增殖,诱导细胞阻滞细胞于G2/M期%Objective To investigated the the effects of EZH2 RNAi on the growth of gastric cancer cell line MKN1.Methods Plasmid pSilence(+)expressing RNAi was transfected into MKNI cells by lipofectamine,we detected the expression of EZH2 in MKN1,MKNl/Silence(+)and MKNl/Silence(-).Then,we examined the proliferation with MTT assay and cycle distribution with flow cytometric assay of MKNI,MKN1/Silence(+)and MKNl/Silence(-).Results Plasmid pSilence(+)and pSilence(-)were transfected into MKN1 cells by lipofectamine,then stablyresistant cells were selected using G418,pSJlence(+)significantly knocked down the EZH2 expression with RT-PCR and Western blot,EZH2-siRNA markedly inhibited MKNI proliferation,Flow cytometric analysis showed that MKN1/pSilence(+)cells cycle distribution was in the G2/M phase(36.08 ± 1.34),higher to the level of MKN1/pSilenec(-)(18.35 ± 0.35)and MKN 1 cells(16.59 ± 0.71),which showed that EZH2 siRNA induced MKN1 cells arrested in the G2/M phases.Conclusion EZH2-siRNA markedly inhibites MKN1 proliferation; EZH2 siRNA induces MKN1 cells arrested in the G2/M phases.
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