The Expression Of MiRNA-26 During ATRA-induced Differentiation Of HL-60 Cells Towards Granulocytes

Background:Acute myeloid leukemia (AML) is a heterogeneous group of diseases characterized by uncontrolled proliferation of clonal neoplastic hematopoietic precursor cells as well as blockage of myeloid differentiation at different stages. Changes in gene, such as chromosomal translocation, rearrangements, point mutation, deletion, DNA methylation, were invovled in the process of leukemogenesis through oncogene activation and tumor suppressor gene inactivation. In rencent years, the study of epigenetic mechanisms becomes increasingly important for cancer research after the successful clinical trail using the demethylating agent azacitidine and its deoxy derivative 5-Aza-2'-deoxycytidine (5-Aza-dC). MicroRNAs (miRNAs) are endogenous 18-25-nucleotide non-coding RNAs that have emerged as a new paradigm of epigenetic regulation both in normal developmental and metabolic processes and in the pathogenesis of human disease including cancer. Emerging evidence demonstrates that miRNAs also play an essential role in differentiation and proliferation of AML. Differentiation therapy with all-trans retinoic acid (ATRA) provides a successful strategy for acute promyelocytic leukemia. Approximately 70%-80% of patients with newly diagnosed APL achieve long-term remission and probably are cured. However, the epigenetic mechanism by which ATRA induces the differentiation of leukemia cells has been largely unknown, especially for the differentiation of acute myeloid leukemia cells, in which the PML-RARa fusion gene expression is negative. Therefore, fluorescence quantitative real-time PCR was used to detect the expression level of miR-26 in acute myeloblastic leukemia cell lines HL-60 induced by ATRA. This setting provides a basis for the regulation of miR-26 in the differentiation process.Objective:To investigate the expression pattern of microRNA-26 (miR-26) in the differentiation of acute myeloblastic leukemia (AML) cell line HL-60 induced by ATRA.Methods:HL-60 cells stimulated by ATRA(1μmol/L) capable of inducing differentiation for different time were used as subjects. The alterations in cell morphology were observed with Wright staining under microscope. The differentiation marker on the cell membrane surface was analyzed by using flow cytometry. The miRNAs expression was verified by using real-time fluorescence quantitative PCR.Results:1) With drug-inducing time prolonged, the differentiation along the neutrophil lineage was proved and the nuclei morphology of the differentiation cells was changed.2) As compared with control group, the differentiating rate induced by ATRA (1μmol/L) at 3d,4d,5d and 6d was (52.39±3.56)%, (77.65±0.24)%,(83.90±1.30)%, and (68.13±1.67)%, (P<0.01), respectively.3) The relative expressions of miR-26A and miR-26B were significantly up-regulated at 5d, and showed remarkable difference (P<0.05).Conclusion:miR-26A and miR-26B are highly expressed in the differentiation of HL-60 cells induced by ATRA, which indicates that miR-26 may be invovled in the regulation process of granulocytic differentiation.