N6-甲基腺苷(m6A)修饰是真核生物中最丰富的可逆甲基化修饰，据报道与多种癌症进展密切相关，包括结直肠癌(CRC)。本研究显示，激活的脂质代谢和糖酵解在CRC的发生和发展中起着重要作用。然而，只有少数研究报道了这种联系的生物学机制。使用Western blot和qRT-PCR测量FTO和ALKBH5的蛋白和mRNA水平。使用CCK-8、集落形成和EdU分析考察FTO和ALKBH5对细胞增殖的影响，使用Transwell分析测试其对细胞迁移和侵袭的影响。采用m6A RNA免疫沉淀(MeRIP)和RNA-seq来探索下游靶基因。使用RIP验证m6A和HK2之间的相互作用。通过裸鼠异种移植模型确定FTO和ALKBH5在体内的功能。在本研究中，发现在高脂环境下，CRC患者和细胞中FTO和ALKBH5的表达显著下调。此外，FTO和ALKBH5的过表达抑制了细胞增殖，相反，FTO和ALKBH5的敲低加速了CRC细胞的恶性生物学行为。FTO和ALKBH5的作用机制涉及到对HK2的共同调控，该酶是糖酵解中的关键酶，是通过RNA测序和MeRIP-seq鉴定的。此外，FTO和ALKBH5的表达降低共同激活了FOXO信号通路，导致CRC细胞的增殖能力增强。作为m6A阅读器，IGF2BP2以m6A依赖方式正调控HK2 mRNA。此外，FTO/ALKBH5的下调增加了METTL3的表达，并降低了METTL14的表达，从而进一步促进CRC的进展。综上所述，本研究揭示了FTO-ALKBH5/IGF2BP2/HK2/FOXO1轴作为CRC异常m6A修饰和糖酵解调控的机制。 © 2023. 中美华人生物学家协会 (SCBA).

N6-methyladenosine (m6A) modification is the most abundant reversible methylation modification in eukaryotes, and it is reportedly closely associated with a variety of cancers progression, including colorectal cancer (CRC). This study showed that activated lipid metabolism and glycolysis play vital roles in the occurrence and development of CRC. However, only a few studies have reported the biological mechanisms underlying this connection.Protein and mRNA levels of FTO and ALKBH5 were measured using western blot and qRT-PCR. The effects of FTO and ALKBH5 on cell proliferation were examined using CCK-8, colony formation, and EdU assays, and the effects on cell migration and invasion were tested using a transwell assay. m6A RNA immunoprecipitation (MeRIP) and RNA-seq was used to explore downstream target gene. RIP was performed to verify the interaction between m6A and HK2. The function of FTO and ALKBH5 in vivo was determined by xenograft in nude mice.In this study, FTO and ALKBH5 were significantly down-regulated in CRC patients and cells both in vivo and in vitro in a high-fat environment. Moreover, FTO and ALKBH5 over-expression hampered cell proliferation both in vitro and in vivo. Conversely, FTO and ALKBH5 knockdown accelerated the malignant biological behaviors of CRC cells. The mechanism of action of FTO and ALKBH5 involves joint regulation of HK2, a key enzyme in glycolysis, which was identified by RNA sequencing and MeRIP-seq. Furthermore, reduced expression of FTO and ALKBH5 jointly activated the FOXO signaling pathway, which led to enhanced proliferation ability in CRC cells. IGF2BP2, as a m6A reader, positively regulated HK2 mRNA in m6A dependent manner. Additionally, down-regulation of FTO/ALKBH5 increased METTL3 and decreased METTL14 levels, further promoting CRC progression.In conclusion, our study revealed the FTO-ALKBH5/IGF2BP2/HK2/FOXO1 axis as a mechanism of aberrant m6A modification and glycolysis regulation in CRC.© 2023. Society of Chinese Bioscientists in America (SCBA).