Hematology (Amsterdam, Netherlands) · 2025
The role of METTL14 in the progression of chronic myeloid leukemia.
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Zhang Jing, Liao Zhi-Hua, Xu Yan-Mei, Li Shu-Qi, Zhong Fang-Min, Zhang Ling, Yao Fang-Yi, Bai Qin, Yao Li-Hua, Huang Bo, Liu Jing, Wang Xiao-Zhong
Аннотация
OBJECTIVES: Chronic myeloid leukemia (CML), a clonal malignant disease arising from the BCR-ABL fusion gene, presents significant therapeutic challenges, particularly in chemotherapy resistance. The role of METTL14, a key m6A methyltransferase, is implicated in cancer biology, but its role in CML remains unclear.
METHODS: Peripheral blood mononuclear cells (PBMCs) and CML cell lines (K562 and K562/G01) were conducted in vitro studies. mRNA levels were quantified by quantitative PCR (qPCR), and protein expressions were assessed by Western Blotting. Cell viability and apoptosis were measured using the CCK-8 and flow cytometry, respectively. Drug resistance was evaluated by determining the half-maximal inhibitory concentration (IC50). m6A levels were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and modification sites were predicted by SRAMP and confirmed with a SELECT detection assay. Gene interactions were validated through luciferase assays.
RESULTS: METTL14 expression was significantly elevated in imatinib-resistant CML patients (P=0.005) and K562/G01 cells (P=0.01), correlating with increased m6A modification levels (P=0.032). Overexpression of METTL14 enhanced m6A methylation, promoted cell proliferation, inhibited apoptosis, and increased imatinib resistance in CML cells. Conversely, METTL14 silencing reduced m6A levels, induced G0/G1 arrest, and enhanced apoptosis (P=0.01). Mechanistically, the luciferase assay results demonstrated that METTL14-mediated m6A modification at the A1001 site of Bcl-x mRNA facilitated HNRNPC-dependent splicing. Consequently, this modification results in shifting Bcl-xS to Bcl-xL and inactivating the BCL-2/BAX/Caspase-3 pathway.
CONCLUSION: METTL14-driven m6A modification regulates the splicing pattern of Bcl-x, and may facilitate the progression of CML. Keywords: CML, METTL14, N6-methyladenine, Bcl-x, Alternative splicing, resistance, imatinib, progression.
METHODS: Peripheral blood mononuclear cells (PBMCs) and CML cell lines (K562 and K562/G01) were conducted in vitro studies. mRNA levels were quantified by quantitative PCR (qPCR), and protein expressions were assessed by Western Blotting. Cell viability and apoptosis were measured using the CCK-8 and flow cytometry, respectively. Drug resistance was evaluated by determining the half-maximal inhibitory concentration (IC50). m6A levels were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and modification sites were predicted by SRAMP and confirmed with a SELECT detection assay. Gene interactions were validated through luciferase assays.
RESULTS: METTL14 expression was significantly elevated in imatinib-resistant CML patients (P=0.005) and K562/G01 cells (P=0.01), correlating with increased m6A modification levels (P=0.032). Overexpression of METTL14 enhanced m6A methylation, promoted cell proliferation, inhibited apoptosis, and increased imatinib resistance in CML cells. Conversely, METTL14 silencing reduced m6A levels, induced G0/G1 arrest, and enhanced apoptosis (P=0.01). Mechanistically, the luciferase assay results demonstrated that METTL14-mediated m6A modification at the A1001 site of Bcl-x mRNA facilitated HNRNPC-dependent splicing. Consequently, this modification results in shifting Bcl-xS to Bcl-xL and inactivating the BCL-2/BAX/Caspase-3 pathway.
CONCLUSION: METTL14-driven m6A modification regulates the splicing pattern of Bcl-x, and may facilitate the progression of CML. Keywords: CML, METTL14, N6-methyladenine, Bcl-x, Alternative splicing, resistance, imatinib, progression.