To investigate the effects and underlying mechanisms of down-regulating m⁶A demethylase fat mass and obesity-associated protein (FTO) on proplatelet formation in the MEG-01 megakaryocytic cells.

①MEG-01 cells were treated with 1 nmol/L phorbol myristate acetate (PMA) (treatment group) or DMSO (control group) for 72 h. FTO expression was measured by Western blotting and RT-qPCR. ② MEG-01 cells were infected with targeted FTO shRNA (knockdown group, sh-FTO) or negative control shRNA (negative control group, sh-NC) viruses. FTO knockdown group and negative control group MEG-1 cells were treated with 1 nmol/L PMA for 72 h, and the protein and mRNA expression levels of FTO were detected by Western blotting and RT-qPCR. Cell cycle, viability and apoptosis were assessed by propidium iodide (PI) DNA staining, CCK-8 assay and Annexin V-FITC/PI double staining and TUNEL staining. The expression of cleaved Caspase-3 protein was determine by Western blotting. Megakaryocyte maturation was assessed by CD41/CD61 staining. Proplatelet formation was observed under bright field and detected by CD61 immunofluorescence assay. The expression of apoptosis-related molecules (Caspase3, BAD, BAK1, BCL2 and MCL1) was detected by RT-qPCR, and the protein change of BCL2 was further verified by Western blotting. The dataset was screened out from the gene expression omnibus (GEO) database, and then analyzed with University of California, Santa Cruz (UCSC) genome browser to compare the methylation sequencing peaks on BCL2 mRNA, and m⁶A methylated RNA immunoprecipitation (m⁶A-RIP) was used to assess the m⁶A methylation levels of BCL2 target gene mRNAs in MEG-01 megakaryocytes. Then, the changes in the m⁶A methylation enrichment level of BCL2 mRNA were observed between the sh-NC group and the sh-FTO group. mRNA stability and ribosome profiling assays were performed to assess translational efficiency of target genes.

①PMA treatment upregulated the expression of FTO at protein (P<0.05) and mRNA (P<0.01) levels. ② FTO shRNA resulted in reduced FTO expression at both mRNA and protein levels (P<0.01). Compared to the negative control group, the FTO knockdown group showed more cells arrested at the G1/S phase [(60.80±1.29)% vs (72.13±1.18)%, P<0.01], significantly reduced cell viability [(1.17±0.03)% vs (0.69±0.05)%, P<0.01], increased Annexin V-FITC/PI positive cells [(12.87±0.83)% vs (17.45±1.58)%, P<0.01], more TUNEL positive cells [(1.03±0.27)% vs (17.49±9.91)%, P<0.01], enhanced protein level of cleaved Caspase-3 (P<0.01), decreased proportion of CD41/CD61 positive cells [(51.63±1.13)% vs (34.08±0.53)%, P<0.01], and less proplatelet formation in MEG-01 megakaryocytes [(26.49±6.73)% vs (13.31±5.97)%, P<0.01]. ③Compared to sh-NC group, the FTO knockdown group had significantly decreased protein and mRNA expression of anti-apoptotic molecule BCL2 (P<0.01). UCSC GEO sequencing data revealed there were m⁶A methylation modification sites on BCL2 mRNA, which was verified through m⁶A-RIP experiment in MEG-01 megakaryocytes. Compared with GAPDH mRNA, BCL2 mRNA exhibited a significantly enriched m⁶A signal (P<0.01). Compared to sh-NC group, a significant increase in m⁶A methylation modification was observed on BCL2 mRNA. BCL2 mRNA stability was significantly decreased, and its translation efficiency significantly was decreased(P<0.01).

m⁶A demethylase FTO rgulates BCL2 mRNA stability and translation efficiency，thereby promoting proplatelet formation in MEG-01 megakaryocytes.