GTP酶Rab25在结直肠癌化疗耐药中的作用研究

The role of GTPase Rab25 in chemotherapy resistance of colorectal cancer

目的研究GTP酶Rab25与结直肠癌化疗耐药的关系。方法 MTT比色法检测细胞的耐药性;全转录组测序技术筛选结直肠癌细胞耐药相关的GTP酶Rab家族基因;基于生物信息学分析Rab25与患者预后以及多药耐药标志物的相关性;实时荧光定量PCR和免疫印记法检测细胞Rab25的RNA表达水平和蛋白水平;流式细胞术检测耐药细胞过表达Rab25前后的周期及凋亡。结果抗癌药物处理,耐药株细胞存活率显著高于亲本株(P <0.01);Rab25、Rab32和Rab38在耐药株里面显著低表达(P <0.001);Rab25高表达的结直肠癌患者总体生存期更长(HR=0.4,P <0.05),Rab25与多药耐药标志物ABCB1、ABCC1和ABCG2表达均呈负相关(P <0.01);与HCT-8相比,耐药株Rab25的RNA和蛋白水平显著降低,耐药株转染Rab25质粒后RNA和蛋白水平表达显著增高(P <0.001);上调Rab25增强耐药细胞对药物的敏感性(P <0.001);上调Rab25增强药物对耐药细胞G0/G1期的阻滞,促进药物对耐药细胞的致凋亡作用(P <0.001)。结论上调Rab25可显著降低结直肠癌耐药细胞株的化疗耐药性。

Objective To study the relationship between GTPase Rab25 and chemotherapy resistance in colorectal cancer. Methods MTT colorimetric method was used to detect the drug resistance of cells. Whole transcriptome sequencing technology was used to screen the GTPase Rab family genes related to the drug resistance of colorectal cancer cells. Bioinformatics was used to analyze the correlation of Rab25 with patient prognosis and multidrug resistance biomarkers. RT-PCR and Western Blot were used to verify the RNA and protein expression level of Rab25 in cells. Flow cytometry was used to detect the cycle and apoptosis of drug-resistant cells before and after the overexpression of Rab25. Results The cell survival rate of drug-resistant strains was significantly higher than that of parental strains treated with anticancer drugs(P < 0.01). Rab25,Rab32,and Rab38 were significantly lower expressed in drug-resistant strains(P < 0.001). Colorectal cancer patients with high expression of Rab25 had a longer overall survival(HR = 0.4,P < 0.05)and Rab25 was negatively correlated with the expression of multidrug resistance markers ABCB1,ABCC1 and ABCG2(P < 0.01). Compared with HCT-8,the RNA and protein level of the drug-resistant strain Rab25 was significantly reduced,and the RNA and protein level of the drug-resistant strain was significantly increased after transfection of the Rab25 plasmid(P < 0.001). Up-regulation of Rab25 enhanced the sensitivity of drug-resistant cells to drugs(P < 0.001). Up-regulation of Rab25 enhanced drug-resistant cell G0/G1 blockade,and promoted drug-induced apoptosis of drug-resistant cells Effect(P < 0.001). Conclusion Up-regulation of Rab25 can significantly reduce the chemotherapy resistance of colorectal cancer drug-resistant cell lines.