ALKBH5 promotes T-cell acute lymphoblastic leukemia growth via m6A-guided epigenetic inhibition of miR-20a-5p.

Abstract

This study investigates the role of ALKBH5-mediated m⁶A demethylation in T-cell acute lymphoblastic leukemia (T-ALL). T-ALL cell lines (HPB-ALL, MOLT4, Jurkat, CCRF-CEM) and human T cells were analyzed. CCRF-CEM and Jurkat cells were transfected with si-ALKBH5, miR-20a-5p-inhibitor, and pcDNA3.1-DDX5. The expression levels of ALKBH5, miR-20a-5p, and DDX5 in these cells were determined using qRT-PCR and Western blotting. Cell viability, proliferation, colony formation, and apoptosis were assessed using CCK-8, EdU staining, colony formation assay, and flow cytometry. mRNA m6A levels were quantified with an m6A RNA methylation detection reagent, and RNA immunoprecipitation was employed to measure the enrichment of DGCR8 and m6A on the primary transcript pri-miR-20a of miR-20a-5p. Dual-luciferase assay confirmed the binding relationship between miR-20a-5p and DDX5. Results showed that ALKBH5 and DDX5 were upregulated in T-ALL tissues and cells, whereas miR-20a-5p was downregulated. Silencing ALKBH5 inhibited T-ALL cell viability, colony formation, and proliferation, while promoting apoptosis. These effects were reversed by miR-20a-5p inhibition or DDX5 overexpression. ALKBH5 reduced the relative m⁶A level in T-ALL cells and decreased miR-20a-5p expression by reducing DGCR8 binding to pri-miR-20a-5p. miR-20a-5p suppressed DDX5 transcription. In conclusion, ALKBH5-mediated m⁶A demethylation decreases DGCR8 binding to pri-miR-20a, thereby repressing miR-20a-5p expression and enhancing DDX5 expression, ultimately inhibiting T-ALL cell apoptosis and promoting proliferation.