METTL3介导的m6A修饰在结直肠癌奥沙利铂耐药中的作用研究

Role of N6-methyladenosine mediated by methyltransferase-like 3 in oxaliplatin resistance of colorectal cancer

摘要:

目的 探究N⁶-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 m⁶A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m⁶A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 m⁶A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m⁶A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。

Abstract:

Objective To investigate the role of N⁶-methyladenosine （m⁶A） 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 m⁶A in HCT116/OXA and parental cells. RNA-seq and qRT-PCR were used to screen the differential regulators of m⁶A. 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 m⁶A 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 METTL3 significantly 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 m⁶A methylation modification may promote the OXA resistance of colorectal cancer by regulating ABC transporter，stem cell pluripotency and TGF-β signaling pathway and other classical drug resistance signaling pathways.