Methylation Status of miRNA Let-7a-3 in Acute Myeloid Leukemia

Abstract

An increasing number of epigenetic deregulation was reported to be associated with acute myeloid leukemia (AML). Hypermethylation of several genes is associated with hematologic malignancies, such as the calcitonin gene, p15INK4B, CEBPA, SOCS1, DAPK, p21Cip/Waf1, the ER gene, SDC4, MDR. MicroRNAs (miRNAs) are endogenous nonconding small RNAs that inhibit the translation of genes whose 3′-UTR are targeted. Many miRNA genes, including let-7a-3, are located in CpG islands, suggesting possible epigenetic regulation of their expression. The let-7a-3 gene has been reported to be heavily methylated in normal human placenta, brain, bone marrow, colon, skin, and lung tissue but unmethylated in some lung cancer samples. In this communication, we used bisulfite sequencing and real-time reverse transcription-PCR to analyze methylation status in 33 CpG sites of the let-7a-3 gene and miRNA expression of let-7a in four myeloid cell lines, HEL, HL60, K562 and U937. All of them showed hypermethylation (93%, 80%, 71% and 90%, respectively) in let-7a-3 promoter region (Figure 1) and displayed little let-7a miRNA expression. To evaluate the significance of let-7a-3 methylation status in patients with AML, 90 patients with de novo AML were evaluated for let-7a-3 methylation status by using combined bisulfite restriction analysis (COBRA) and confirmation with bisulfite sequencing. Most AML patients were noted to have hypermethylation in the promoter region of let-7a-3 gene. We divided these patients into three groups: demethylated (n=6), hypomethylated (n=11) and hypermethylated (n=73). Correlation of let-7a-3 methylation with biological and clinical features was evaluated. We found that the methylation status was not associated with patient age at diagnosis, sex, FAB classification and cytogenetic change. Further sequential study revealed that no association was found between let-7a-3 methylation and disease status at either complete remission or relapse stage. We previously demonstrated that 25% AML patients showed hypermethylation in distant region of CEBPA promoter and the methylation status correlated with disease status. In this study, we found that up to 81% AML patients showed let-7a-3 methylation. In the meanwhile, further analysis revealed inverse association between CEBPA and let-7a-3 methylation (p=0.007). It has be widely known that CpG islands hypermethylation of tumor-suppressor genes is a common alteration in cancer cells, leading to the transcriptional inactivation of these genes and normal cellular malfunctions. At the same time, it is also known that the genome of the cancer cell undergoes global hypomethylation at repetitive sequences, leading to genome fragility and tumorigenesis. CEBPA is widely known to be tumor suppressor gene, and let-7a-3 was previously identified as with oncogenic function in lung adenocarcinomas. Consistent to the results shown in lung adenocarcinomas, our results indicate that the miRNA let-7a might play an oncogenic function in AML. [Display omitted]