Abstract: Objective To investigate the molecular mechanism by which methyltransferase-like 3 (METTL3) promotes megakaryocyte differentiation via m⁶A modification-mediated regulation of integrin β3 (ITGB3) expression. Methods Human megakaryoblastic leukemia cells (MEG-01) treated with 20 nM phorbol myristate acetate (PMA) were used to establish a differentiation model. Baseline METTL3 expression in MEG-01 cells and hematopoietic stem cell (HSC) differentiation models was detected by Western blot (WB) and quantitative real-time PCR (RT-qPCR). Subsequently, lentivirus-mediated shRNA (shMETTL3) was used to transfect the MEG-01 megakaryocytic cell line. Knockdown efficiency was detected by WB and RT-qPCR. The expression levels of megakaryocytic surface differentiation markers CD41a and CD61 were measured by flow cytometry. Transcriptome sequencing was applied to identify downstream targets of METTL3, and their expression levels were validated by RT-qPCR and WB. Furthermore, in METTL3-overexpressing cells, the expression levels of METTL3 were detected by RT-qPCR and WB, and CD41a and CD61 were analyzed by flow cytometry. Additionally, METTL3 was re-expressed in METTL3-silenced MEG-01 cells, and its expression was assessed by RT-qPCR and WB, while the levels of CD41a and CD61 were detected by flow cytometry. Finally, bioinformatic prediction (SRAMP) was employed to identify potential m⁶A modification sites on ITGB3 mRNA, and m⁶A-RIP combined with Me-PCR was performed to examine the enrichment of m⁶A signals. Results METTL3 was expressed at mRNA and protein levels in MEG-01 cells and HSC-derived megakaryocytes.shMETTL3 significantly reduced METTL3 mRNA and protein (P<0.001). shMETTL3 decreased CD41a and CD61 surface expression versus the Scramble control (P<0.001). Transcriptomics identified ITGB3 (encoding CD61) as a key target; its mRNA and protein decreased in shMETTL3 cells (P<0.05). METTL3-OE upregulated CD41a and CD61; METTL3 rescue restored their expression (P<0.01). m⁶A-RIP/Me-PCR confirmed: (1) Higher m⁶A enrichment on ITGB3 versus GAPDH mRNA (P<0.001); (2) Reduced m⁶A modification on ITGB3 in shMETTL3 cells (P<0.001). Conclusion Downregulation of the RNA methyltransferase METTL3 reduces m⁶A modification on ITGB3 mRNA, thereby suppressing ITGB3 expression and impairing megakaryocyte differentiation and platelet production.

Key words: METTL3, m⁶A methylation, Platelet production, Megakaryocyte differentiation, ITGB3, CD61