RIPK2上调MGMT调节结肠癌细胞伊立替康耐药的机制探讨

Mechanism of RIPK2 up-regulating MGMT to regulate irinotecan resistance in colon cancer cells

目的探讨受体相互作用丝氨酸苏氨酸激酶2(RIPK2)过表达在结肠癌细胞伊立替康耐药中的作用及相关机制。方法构建HCT116细胞伊立替康耐药细胞HCT116/DR。MTT法检测细胞增殖能力;蛋白印记实验检测细胞RIPK2和O-6-甲基鸟嘌呤DNA甲基转移酶(MGMT)蛋白表达;慢病毒转染法外源性过表达HCT116细胞RIPK2;结晶紫染色法检测伊立替康对细胞的毒性作用。结果HCT116/DR细胞构建成功,耐药指数为4.93。与HCT116细胞相比,RIPK2和MGMT蛋白表达在HCT116/DR细胞中表达明显升高(P<0.01)。RIPK2过表达显著诱导HCT116细胞MGMT蛋白表达(P<0.01)。RIPK2过表达明显抵抗伊立替康诱导的HCT116细胞贴壁存活率降低(P<0.01)。HCT116细胞RIPK2过表达后耐药能力明显增强,耐药指数为4.57。结论RIPK2可能通过上调MGMT蛋白表达参与结肠癌细胞伊立替康耐药。

Objective To investigate the role of overexpression of receptor-interacting serine-threonine kinase 2(RIPK2)in irinotecan resistance in colon cancer cells and the related mechanisms.Methods Irinotecan-resistant cells were constructed with colon cancer cells HCT116 and were named as HCT116/DR.Cell proliferation ability was measured by MTT assay.Western blot analysis was used to detect RIPK2 and O-6-methylguanine DNA methyltransferase(MGMT)protein expression in the cells.Lentiviral transfection was performed to exogenously overexpress RIPK2 in HCT116 cells.The crystal violet staining method was used to detect irinotecan-induced cyto-toxicity.Results HCT116/DR cells were successfully constructed with a drug resistance index of 4.93.RIPK2 and MGMT protein expression was significantly increased in HCT116/DR cells(P<0.01)compared to that in HCT116 cells.RIPK2 overexpression significantly induced the MGMT protein expression in HCT116 cells(P<0.01)and significantly conferred the decrease of the survival rate of irinotecan-induced adherent HCT116 cell(P<0.01).RIPK2 overexpression significantly enhanced the drug resistance of HCT116 cells,resulting in a drug resistance index of 4.57.Conclusion RIPK2 contributes to irinotecan resistance in colon cancer cells by up-regulating MGMT protein expression.