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目的下调EZH2的表达,探讨EZH2在胃癌细胞株MKNl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MKNl中,G418选择培养,建立EZH2低表达的胃癌细胞系MKNl/Silence(+);RT-PCR和Western blot检测EZH2在MKNl、MKNl/Silence(+)和MKNl/Silence(-)细胞表达,MTT法检测细胞增殖的活性,流式细胞观察其对MKNl细胞周期的影响。结果构建成功稳定表达EZH2 siRNA细胞MKNl/Silence(+):EZH2在MKNl/Silenc(+)的mRNA和蛋白的表达明显下降,MKNl/Silence(+)细胞增殖明显抑制。在细胞周期试验中,MKNl/Silence(+)细胞G2/M期(36.08±1.34)比MKNl(16.59±0.71)、空载体MKNI/Silence(-)(18.35±0.35)显著增高(P<0.01),G0/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 investigate the role of EZH2 in the proliferation and cell cycle of gastric cancer cell line MKN1 and the role of EZH2 in the development and progression of gastric cancer. Methods EZH2 siRNA expression vector pSilencer 2.1-U6 (+) was transfected into human gastric cancer cell line MKN1 by Lipofect AUINE 2000. EZH2 low expression gastric cancer cell line MKN1 / Silence (+) was established by G418 selection. RT-PCR and Western The expression of EZH2 in MKN1, MKN1 / Silence (+) and MKN1 / Silence (-) cells was detected by Western blot. The proliferation of MKN1 / Silence (-) cells was detected by MTT assay. Results The expression of MKN1 / Silence (+): EZH2 mRNA and protein in MKN1 / Silenc (+) cells decreased and the proliferation of MKN1 / Silence (+) cells was significantly inhibited. In the cell cycle assay, the G2 / M phase of MKN1 / Silence (+) cells was significantly higher (36.08 ± 1.34) than MKN1 (16.59 ± 0.71) and empty vector MKNI / Silence (- , The number of G0 / G1 phase cells was significantly decreased (P <0.01), while there was no significant change in S phase cells. The cells were arrested in G2 / M phase by EZH2 siRNA. Conclusion The down-regulation of EZH2 expression significantly blocked the proliferation of MKN1 cells and induced cell arrest in G2 / M phase