Abstract <p>Colorectal cancer (CRC) is a prevalent malignant tumor witha high death rate globally, whose metastasis is the primary causeof treatment failure and death. Recent studies indicate that epigeneticmodifications (e.g., N⁶-methyladenosine[m⁶A] RNA modification) play a pivotalrole in tumor progression. Specifically, methyltransferase-like3 (METTL3)-mediated m⁶A modificationinfluences cell death mechanisms and mitochondrial function by regulatingdownstream gene expression, thereby promoting CRC invasion and metastasis.However, its role in ferroptosis and metabolic regulation warrants furtherinvestigation. In this study, the expression levels of METTL3 and5'-AMP-activated protein kinase catalytic subunit alpha-2 (PRKAA2)in CRC were analyzed using data from The Cancer Genome Atlas (TCGA)-COADdataset and validated in CRC cells by quantitative real-time polymerasechain reaction (RT-qPCR) and Western blot. The results showed thatMETTL3 was highly expressed in both CRC tissues and cell lines.The results demonstrated that METTL3 overexpression significantlyreduced the levels of core ferroptosis indicators (Fe²⁺,MDA, and cellular/mitochondrial ROS) and increased the mitochondrialmembrane potential in HCT15 cells. Furthermore, Western blot analysisrevealed that METTL3 overexpression upregulated the protein expressionof GPx4, HSP60, and MFN2 in HCT15 cells. Transwell assays indicatedthat METTL3 overexpression enhanced the migration and invasion of HCT15cells. PRKAA2 is downregulated in CRC tissues and cell lines. Overexpressionof PRKAA2 reversed the inhibitory effects of METTL3 overexpressionon ferroptosis and mitochondrial dysfunction and attenuated themigration and invasion capabilities of HCT15 cells. METTL3 overexpressionincreased the m⁶A modification levelof PRKAA2, decreased its mRNA stability, and subsequently significantlyinhibited the mRNA and protein expression of PRKAA2. Finally, alung metastasis mouse model was established by tail vein injectionof HCT15 cells. In vivo experiments showed that METTL3 overexpressionpromoted the formation of lung metastatic foci, increased the numberof pulmonary nodules, reduced Fe²⁺, MDA,and ROS levels in lung tissues, and upregulated the expression ofGPx4, HSP60, and MFN2. Conversely, PRKAA2 overexpression effectively reversedthe in vivo effects of METTL3 overexpression and inhibited CRC lungmetastasis. In conclusion, both in vivo and in vitro experiments demonstratethat METTL3 suppresses PRKAA2 expression by reducing its mRNA stabilitythrough m⁶A modification. This revealsa novel mechanism by which METTL3 targets PRKAA2 via m⁶Amodification to regulate ferroptosis and mitochondrial function,thereby facilitating tumor metastasis in CRC from the perspectiveof intracellular molecular regulation.</p>