miRNA-125b经p53通路调控Alisertib耐药细胞代谢的机制研究

Targeting microRNA-125b inhibited the metastasis of Alisertib resistance cells through mediating p53 pathway

目的探讨Alisertib耐药的分子机制并寻找Alisertib耐药的潜在靶点。方法用Alisertib连续处理结肠癌细胞系建立耐药细胞株并命名为HCT-8-7T细胞,将HCT-8-7T细胞及人结肠癌细胞系HCT-8接种至免疫缺陷小鼠体内,观察小鼠其他器官转移瘤的情况。采用细胞克隆实验及Transwell迁移实验检测细胞的增殖及迁移能力,采用Seahorse分析仪检测细胞糖酵解及谷氨酰胺代谢能力的差异,Western blot和实时荧光定量聚合酶链反应检测糖酵解及上皮-间质转化(EMT)标志物相关蛋白表达水平及mRNA水平。结果接种HCT-8-7T细胞的小鼠肝脏、肺脏、肾、卵巢等转移灶数量多于接种HCT-8细胞的小鼠(均P<0.05)。HCT-8-7T细胞腺嘌呤核苷三磷酸水平[(0.10±0.01)mmol/L]、糖酵解水平[(81.77±8.21)mpH/min]及糖酵解能力[(55.50±3.48)mpH/min]均高于HCT-8细胞[分别为(0.04±0.01)mmol/L、(27.77±2.55)mpH/min和(14.00±1.19)mpH/min,均P<0.05]。HCT-8-7T细胞中p53蛋白表达水平及mRNA表达水平低于HCT-8细胞(均P<0.05)。HCT-8-7T细胞中miR-125b表达水平(2.21±0.12)高于HCT-8细胞(1.00±0.00,P<0.001)。在HCT-8-7T细胞中,过表达p53导致细胞克隆数[(162.00±24.00)个]及细胞迁移率[(18.53±5.67)%]低于空白对照组[分别为(274.70±40.50)个和(100.00±29.06)%,均P<0.05]。miR-125b mimic组HCT-8-7T细胞糖酵解水平[(25.28±9.51)mpH/min]低于空白对照组[(54.38±12.70)mpH/min,P<0.05]。与空白对照组HCT-8-7T细胞比较,miR-125b mimic组HCT-8-7T细胞中p53和β-catenin蛋白水平升高,PFK1和HK1蛋白水平降低(均P<0.05)。结论miR-125b沉默p53是导致Alisertib耐药的机制之一,靶向miR-125b是克服Alisertib耐药的潜在可用之策。

Objective To clarify the mechanisms involvement in Alisertib-resistant colorectal cells and explore a potential target to overcome Alisertib-resistance.Methods Drug-resistant colon cancer cell line(named as HCT-8-7T cells)was established and transplanted into immunodeficient mice.The metastasis in vivo were observed.Proliferation and migration of HCT-8-7T cells and their parental cells were assessed by colony formation and Transwell assay,respectively.Glycolytic capacity and glutamine metabolism of cells were analyzed by metabolism assays.The protein and mRNA levels of critical factors which are involved in mediating glycolysis and epithelial-mesenchymal transition(EMT)were examined by western blot and reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR),respectively.Results In comparison with the mice transplanted with HCT-8 cells,which were survival with limited metastatic tumor cells in organs,aggressive metastases were observed in liver,lung,kidney and ovary of HCT-8-7T transplanted mice(P<0.05).The levels of ATP[(0.10±0.01)mmol/L],glycolysis[(81.77±8.21)mpH/min]and the capacity of glycolysis[(55.50±3.48)mpH/min]in HCT-8-7T cells were higher than those of HCT-8 cells[(0.04±0.01)mmol/L,(27.77±2.55)mpH/min and(14.00±1.19)mpH/min,respectively,P<0.05].Meanwhile,the levels of p53 protein and mRNA in HCT-8-7T cells were potently decreased as compared to that in HCT-8 cells(P<0.05).However,the level of miRNA-125b(2.21±0.12)in HCT-8-7T cells was significantly elevated as compared to that in HCT-8 cells(1.00±0.00,P<0.001).In HCT-8-7T cells,forced-expression of p53 reduced the colon number(162.00±24.00)and the migration[(18.53±5.67)%]as compared with those in cells transfected with control vector[274.70±40.50 and(100.00±29.06)%,P<0.05,respectively].Similarly,miR-125b mimic decreased the glycolysis[(25.28±9.51)mpH/min]in HCT-8-7T cells as compared with that[(54.38±12.70)mpH/min,P=0.003]in HCT-8-7T cells transfected with control.Meanwhile,in comparison with control transfected HCT-8-7T cells,miR-125b mimic also significantly led to an increase in the levels of p53 andβ-catenin,in parallel with a decrease in the levels of PFK1 and HK1 in HCT-8-7T cells(P<0.05).Conclusions Silencing of p53 by miR-125b could be one of the mechanisms that contributes to Alisertib resistance.Targeting miR-125b could be a strategy to overcome Alisertib resistance.