Supplementary Materials Appendix EMBJ-39-e103181-s001. we’ve identified FOXO3 as a key downstream target of METTL3, with m6A modification of the FOXO3 mRNA 3\untranslated region Calcineurin Autoinhibitory Peptide increasing its stability through a YTHDF1\dependent mechanism. Analysis of clinical samples furthermore showed that METTL3 and FOXO3 levels are tightly correlated in HCC patients. In mouse xenograft models, METTL3 depletion significantly enhances sorafenib resistance of HCC by abolishing the identified METTL3\mediated FOXO3 mRNA stabilization, and overexpression of FOXO3 restores m6A\dependent sorafenib sensitivity. Collectively, our work reveals a critical function for METTL3\mediated m6A modification in the hypoxic tumor microenvironment and identifies FOXO3 as an important target of m6A modification in the resistance of HCC to sorafenib therapy. and experiments. Taken together, our study reveals a connection of m6A modification, sorafenib resistance, and autophagy under hypoxia, and provides insights into the multiple molecular mechanisms of sorafenib resistance in HCC, as well as expanding the understanding of therapy resistance. Results Down\regulation of METTL3 in sorafenib\resistant HCCs To investigate the molecular mechanism of sorafenib resistance in HCC, we obtained liver tumors with acquired sorafenib resistance (by exposing the cells with sorafenib at 5% of IC concentration for 3?days and gradually increased the concentration by 5% of IC until reaching the IC50 concentration. We confirmed the acquired resistance of these HepG2 cells toward sorafenib by comparing to the Calcineurin Autoinhibitory Peptide na?ve HepG\2 cells (Fig?1G). Importantly, we found that METTL3 was markedly reduced in sorafenib\resistant HepG\2 cells with consistently decreased global RNA m6A level (Fig?1H and I), indicating a potential role of METTL3 in mediating resistance toward sorafenib in HCC cells. Moreover, we noticed that hypoxic condition extensively existed in HCC, as we observed an elevated expression level of HIF\1 in liver tumors in comparison with non\tumor liver tissues (Appendix?Fig S1C and D). We established a HCC subcutaneous tumor model in nude mice with SMMC\7721 cells and found that 80% of tumors exhibited a higher level of hypoxia (Appendix?Fig S1E and F). We also performed an orthotopic liver tumor xenograft with Bel\7402 cells to validate the hypoxic condition in HCC. Higher levels of HIF\1 were detected in tumor tissue compared to non\tumor tissue (Appendix?Fig S1G and G1), indicating oxygen deprivation was a Calcineurin Autoinhibitory Peptide common feature of HCC. To examine the role of METTL3 in HCC, our following functional experiments were conducted at low oxygen levels (1% O2) to mimic the intratumoral microenvironment in HCC. Knockdown of METTL3 enhanced sorafenib resistance in HCC To evaluate whether the global m6A level is related to tumorigenesis in liver cancer, we generated a series of various expression levels of METTL3 in the normal liver cell line WRL68 (Appendix?Fig S2A and B). The normal liver cells formed an enlarged colony after silencing METTL3, but had no meaningful change after overexpressing METTL3 (Fig?2A and B). Neither METTL3 knockdown nor METTL3 overexpression in WRL68 cells induced sensitivity toward sorafenib treatment, indicating that sorafenib was not toxic to normal liver cells regardless of various levels of METTL3 expression (Appendix?Fig S2C). However, knockdown of METTL3 in WRL68 cells moderately promoted cell growth, while overexpression of METTL3 in WRL68 Rabbit Polyclonal to KAP1 cells had no effect in a cell proliferation assay and cell number counting (Appendix?Fig S2D and E). To further explore the role Calcineurin Autoinhibitory Peptide of the m6A modification in HCC toward sorafenib resistance, we constructed six Calcineurin Autoinhibitory Peptide stable METTL3\depeleted HCC cell lines (SMMC\7721 #sh1 #sh2, Bel\7402 #sh1 #sh2,.