Liu Jiazhuo, Zhang Xin, Liao Yi, Zhang Chunlan, Peng Leiwen
Department of Hematology, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, China.
Department of Laboratory Medicine, West China Second University Hospital, Sichuan University, Chengdu, Sichuan, 610041, China.
Exp Cell Res. 2025 Jan 15;444(2):114293. doi: 10.1016/j.yexcr.2024.114293. Epub 2024 Oct 22.
This study investigates the role of ALKBH5-mediated mA 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 mA 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 mA 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.
本研究调查了ALKBH5介导的m⁶A去甲基化在T细胞急性淋巴细胞白血病（T-ALL）中的作用。对T-ALL细胞系（HPB-ALL、MOLT4、Jurkat、CCRF-CEM）和人T细胞进行了分析。用si-ALKBH5、miR-20a-5p抑制剂和pcDNA3.1-DDX5转染CCRF-CEM和Jurkat细胞。使用qRT-PCR和蛋白质免疫印迹法测定这些细胞中ALKBH5、miR-20a-因子水平。使用CCK-8、EdU染色、集落形成试验和流式细胞术评估细胞活力、增殖、集落形成和凋亡。用m⁶A RNA甲基化检测试剂定量mRNA m⁶A水平，并采用RNA免疫沉淀法测量DGCR8和m⁶A在miR-20a-5p的初级转录本pri-miR-20a上的富集情况。双荧光素酶报告基因检测证实了miR-20a-5p与DDX5之间的结合关系。结果显示，ALKBH5和DDX5在T-ALL组织和细胞中上调，而miR-20a-5p下调。沉默ALKBH5可抑制T-ALL细胞活力、集落形成和增殖，同时促进凋亡。miR-20a-5p抑制或DDX5过表达可逆转这些作用。ALKBH5降低了T-ALL细胞中的相对m⁶A水平，并通过减少DGCR8与pri-miR-20a-5p的结合来降低miR-20a-5p的表达。miR-20a-5p抑制DDX5转录。总之，ALKBH5介导的m⁶A去甲基化减少了DGCR8与pri-miR-20a的结合，从而抑制miR-20a-5p表达并增强DDX5表达，最终抑制T-ALL细胞凋亡并促进增殖。
