METTL3 enhances pancreatic ductal adenocarcinoma progression and gemcitabine resistance through modifying DDX23 mRNA N6 adenosine methylation

Abstract

Abstract The aim of the present study was to clarify the mechanism of how METTL3 regulated pancreatic ductal adenocarcinoma (PDAC) progression by m6A modification of its downstream target mRNA and signaling pathway. Immunoblotting and qRT-PCR assays was employed to determine the expression levels of METTL3. In situ fluorescence hybridization was conducted to localize the cellular distribution of METTL3 and DEAD-box helicase 23 (DDX23). CCK8, colony formation, EDU incorporation, TUNEL, wound healing and Transwell assays were carried out accordingly to study the viability, proliferation, apoptosis, and mobility of cells under different treatments in vitro. Xenograft and animal lung metastasis experiments were also conducted to study the functional role of METTL3 or DDX23 on tumor growth and lung metastasis in vivo. MeRIP-qPCR and bioinformatical analyses were used to obtain the potential direct targets of METTL3. It was shown that m6A methyltransferase METTL3 was upregulated in PDAC tissues with gemcitabine resistance, and its knockdown sensitized pancreatic cancer cells to chemotherapy. Furthermore, silencing METTL3 remarkably reduced pancreatic cancer cell proliferation, migration, and invasion both in vitro and in vivo. Mechanistically, validation experiments confirmed that DDX23 mRNA was a direct target of METTL3 in YTHDF1-dependent manner. Additionally, DDX23 silence resulted in the suppression of pancreatic cancer cell malignancy and PIAK/Akt signaling inactivation. Strikingly, rescuse experiments demonstrated the inhibitive effects of METTL3 silence on cell phenotypes and gemcitabine resistance were partially reversed by forcibly expressed DDX23. In summary, METTL3 promotes PDAC progression and gemcitabine resistance by modifying DDX23 mRNA m6A methylation and enhancing PI3K/Akt signaling activation. Our findings establish a potential tumor promotive and chemo-resistant role for METTL3/DDX23 axis in PDAC.