METTL3介导的m^(6)A修饰在结直肠癌奥沙利铂耐药中的作用研究

Role of N^(6)-methyladenosine mediated by methyltransferase-like 3in oxaliplatin resistance of colorectal cancer

目的:探究N^(6)-甲基腺嘌呤(N6-methyladenosine,m6A)调控因子甲基转移酶样蛋白3(methyltransferase-like 3,METTL3)在结直肠癌奥沙利铂(oxaliplatin,OXA)耐药中的作用。方法:采用浓度梯度法构建OXA耐药细胞株HCT116/OXA,CCK-8分析其耐药性;采用Dot blot及m6A RNA甲基化定量ELISA实验检测细胞RNA m^(6)A水平,RNA-seq及qRT-PCR筛选差异m6A调控因子,Western blot验证METTL3表达水平;利用腺病毒感染技术在HCT116细胞过表达METTL3,CCK-8检测各组细胞的增殖活性;通过对GEO数据库中HCT116细胞敲除METTL3的MeRIP-seq和RNA-seq数据与耐药细胞RNA-seq联合分析,筛选耐药相关的METTL3介导m^(6)A修饰调控的下游靶基因及信号通路;TCGA数据库分析METTL3与患者预后的相关性。结果:HCT116/OXA细胞对OXA的耐药性明显高于其亲代细胞(P<0.01);与HCT116相比,耐药细胞的RNA m^(6)A水平异常上调(P<0.01),且伴随METTL3的mRNA和蛋白水平升高;过表达METTL3明显增强HCT116细胞对OXA的耐药性(P<0.01);多组学数据联合分析筛选的METTL3介导m6A修饰调控的与耐药相关的关键基因主要富集在ABC转运蛋白、干细胞多能性调节信号通路、TGF-β信号通路等;METTL3高表达与结直肠癌患者的不良预后明显相关(P<0.01)。结论:METTL3介导的m^(6)A修饰可能通过调节ABC转运蛋白、调节干细胞多能性的信号通路、TGF-β信号通路等经典耐药信号通路促进结直肠癌OXA耐药。

Objective:To investigate the role of N^(6)-methyladenosine(m^(6)A)mediated by methyltransferase-like 3(METTL3)in oxaliplatin(OXA)resistance of colorectal cancer.Methods:Concentration gradient induction method was used to establish OXA resistant cell line HCT116/OXA,and CCK-8 was used to detect the drug resistance of cells. Dot blot and ELISA were used to analyze the expression level of m^(6)A in HCT116/OXA and parental cells. RNA-seq and qRT-PCR were used to screen the differential regulators of m^(6)A. Western blot was used to detect the expression level of METTL3. METTL3 was overexpressed in HCT116 by adenovirus infection technique,and the proliferation activity of each group was detected by CCK-8. The key genes and signal pathways of m^(6)A modification and regulation mediated by METTL3 related to drug resistance were screened through the combined analysis of MeRIP-seq and RNA-seq data of METTL3 knocked out by HCT116 cells in GEO database and RNA-seq data of drug-resistant cells. The correlation between METTL3 and prognosis was analyzed by TCGA database.Results:The resistance of HCT116/OXA cells to OXA was significantly higher than that of their parents(P<0.01). Compared with HCT116 cells,the level of RNA m6A modification in HCT116/OXA cells was significantly up-regulated(P<0.01)and the expression of METTL3 was significantly increased. Overexpression of METTL3significantly enhanced the resistance of HCT116 cells to OXA(P<0.01). Differential genes selected from multiomics data conjoint analysis,related to drug resistance and regulated by METTL3-mediated m6A modification had significant correlation to ABC transporters,stem cell pluripotency,TGF-β signaling pathway and other signaling pathways. The high expression of METTL3 was significantly correlated with the poor prognosis of colorectal cancer patients(P<0.01).Conclusion:METTL3 mediated m^(6)A methylation modificalating ABC transporter,stem cell pluripotency and TGF-β signaling pathway and other classical drug resistance signaling pathways.