DNA methylation-associated dysregulation of ANGPTL4 and TUBB2A in acute myeloid leukemia
Keywords:
Angiopoietin-like 4, beta-Tubulin, DNA Methylation, acute myeloid leukemia, Bisulfite sequencing, TUBB2A.Abstract
Acute myeloid leukemia (AML) is a clonal hematological malignancy marked by genetic and epigenetic alterations. Epigenetic modifications, such as DNA hypermethylation and histone changes, may drive the silencing of tumor suppressor genes (TSGs), a key feature of AML pathogenesis. Angiopoietin-like 4 (ANGPTL4) and tubulin Beta 2A (TUBB2A) genes were identified through transcriptomic analysis of Kasumi-1, an AML cell line, following treatments with epigenetic modulators: Trichostatin A (TSA) and 5-Azacytidine (5-Aza). ANGPTL4, involved in lipid metabolism, promotes tumor growth, metastasis, and angiogenesis, while TUBB2A, part of the microtubule network, is linked to cancer when dysregulated. This study investigates the expression and methylation status of ANGPTL4 and TUBB2A in AML patients, and examines the expression of ANGPTL4, TUBB2A, and histone H3 and H4 proteins in the Kasumi-1 cells treated with a specific agent. Quantitative real-time PCR (qPCR) was performed in 52 de novo AML patients with varying cytogenetic abnormalities. Bisulfite sequencing PCR (BSP) was performed in a representative subset of patients (n = 12). Protein expression of ANGPTL4, TUBB2A, and histone H3 and H4 was analysed by Western blot. The results showed reduced expression of ANGPTL4 in 98.0% of AML patients (median <0.001) and TUBB2A in 73.1% (range 0.33–0.77), with most cases of downregulated TUBB2A found in cytogenetically normal (CN) patients. Bisulfite sequencing revealed fully methylated CpG regions in 96.1% of ANGPTL4 and 54.9% of TUBB2A. Western blot analysis showed the upregulation of ANGPTL4 and TUBB2A proteins, underscoring their functional relevance. Additionally, increased levels of core histones H3 and H4 following TSA treatment suggested an upregulation of global histone gene expression in response to HDAC inhibition. These findings indicate that the underexpression of ANGPTL4 and TUBB2A in AML patients may be a consequence of aberrant hypermethylation of their promoter regions. The downregulation of these genes could support the persistence of leukemic stem cells. Further study on the epigenetic mechanisms underlying the downregulation of ANGPTL4 and TUBB2A in AML is recommended.
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