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本实验主要研究哺乳动物雷帕霉素靶蛋白(mTOR)信号通路与DNA甲基化在人胃癌细胞生存活力、细胞周期、相关基因及蛋白表达方面的相互作用.分别单独或联合DNA甲基化酶抑制剂5-氮杂-2′-脱氧胞苷(5-aza-dC)、mTOR抑制剂雷帕霉素(rapamycin,RAPA)和PI3K抑制剂LY294002干预人胃癌MKN45和SGC7901细胞,以MTT检测细胞生存活力,流式细胞术检测细胞周期,real-timePCR检测PTEN,p27Kip1基因表达情况,亚硫酸氢盐修饰后测序检测DNA甲基化改变,蛋白免疫印迹检测相关蛋白表达情况.结果发现单独应用5-aza-dC对胃癌细胞的抑制作用不明显,当其联合mTOR信号通路抑制剂时则显著抑制胃癌细胞生长(P<0.01);抑制mTOR信号通路可增强5-aza-dC使细胞阻滞在G2期的作用;联合用药还能提高抑癌基因PTEN,p27Kip1的表达,但不影响DNA甲基化状态;LY294002及RAPA使Akt,p70S6K及4E-BP1磷酸化表达显著下降(P<0.01),但5-aza-dC并不增强这一效应.以上研究提示mTOR信号通路抑制剂可间接促进DNA甲基化酶抑制剂对胃癌细胞的抑制作用,为肿瘤的治疗提供新思路.
In this study, the interaction between mammalian target of rapamycin (mTOR) signaling pathway and DNA methylation in human gastric cancer cell viability, cell cycle, related genes and protein expression were studied in this study.Methods: Single or combined DNA methylation Human gastric cancer MKN45 and SGC7901 cells were treated with 5-aza-dC, rapamycin (RAPA) and PI3K inhibitor LY294002, respectively. MTT assay The cell viability was measured by flow cytometry, the expression of PTEN and p27Kip1 was detected by real-time PCR, DNA methylation was detected by bisulfite modified sequencing, and protein expression was detected by Western blotting. 5-aza-dC had no obvious inhibitory effect on gastric cancer cells. When combined with mTOR signaling pathway inhibitor, 5-aza-dC significantly inhibited the growth of gastric cancer cells (P <0.01). Inhibition of mTOR signaling pathway enhanced 5-aza-dC cell block (P <0.01) .Conclusion: LY294002 and RAPA can significantly reduce the phosphorylation of Akt, p70S6K and 4E-BP1 (P <0.01) , But 5-aza-dC is not enhanced Effect a. Above studies suggest that mTOR signaling pathway inhibitor may indirectly contribute to the inhibition of DNA methylation inhibitor on gastric cancer cell, to provide a new method for the treatment of tumors.