Preliminary study on the role of miR-148a and DNMT1 in the pathogenesis of acute myeloid leukemia

MicroRNA (miR)-148a is differentially expressed in numerous malignant tumors and it was identified to regulate tumor growth, cell proliferation, apoptosis, angiogenesis and drug resistance via the regulation of the expression levels of its target genes. However, the biological function of miR-148a in acute myeloid leukemia (AML) and its molecular mechanisms of action remain unclear. In the present study, the expression levels of miR-148a and DNA methyltransferase 1 (DNMT1) were detected using reverse transcription-quantitative polymerase chain reaction (PCR) and western blotting. Methylation-specific PCR was used to detect the methylation levels in the miR-148a promoter. The effects of miR-148a on cell proliferation and apoptosis were assessed by Cell Counting kit-8 or flow cytometry assays, respectively. A dual-luciferase reporter assay was performed to investigate the association between miR-148a and DNMT1. Patients with AML exhibited an increased expression level of miR-148a, whereas the expression level of DNMT1 was identified to be decreased compared with healthy control subjects. In AML cell lines, the methylation state of miR-148 promoter was significantly increased compared with normal cells. Following knockdown of DNMT1 in U937 cells, the expression level of miR-148a increased significantly, whereas the methylation level of the miR-148a promoter decreased. The mRNA and protein expression levels of DNMT1 decreased following transfection with miR-148a mimics in U937 cells. Conversely, transfection with miR-148a inhibitor in Kasumi-1 cells led to an increase in the expression level of DNMT. Dual-luciferase reporter assays suggested that DNMT1 was one of the direct target genes of miR-148a. Overexpression of miR-148a inhibited cell proliferation and promoted apoptosis. Inhibition of DNMT1 led to a decreased methylation level of the 5-cytosine-phosphate-guanine-3 islands in the miR-148a promoter, thus increasing the expression level of miR-148a. DNMT1 was identified to be a downstream target of miR-148a, and was negatively regulated by miR-148a in AML cell lines, suggesting that miR-148a and DNMT1 form a mutual negative feedback loop.