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

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.