Negative regulation of microRNA-9 by ecotropic viral integration site1 involved in the pathogenesis of acute myeloid leukemia

Abstract

Objective To investigate the regulation mechanism of microRNA-9(miR-9)by ecotropic viral integration site1(EVI1)its impact on proliferation of AML cells and its role in the pathogenesis of myelogenous leukemia. Methods EVI1 was forced to express in Uocm1 cell lines by murine stem cell virus-EVI1(MSCV-EVI1) plasmid infection.EVI1 overexpressed Uocm1 cells were then treated with 0.1 μmol/L 5-aza-2′-deoxycytidine (5-AZA) dissolved in dimethyl sulfoxide (DMSO). The methylation level of miR-9 promoter was tested by DNA bisulfite sequencing technology.The cell cycle was observed by flow cytometry (FCM). The proliferation ability of the cells was detected by the colony forming assay in semi-solid Methylcellulose medium culture. Results EVI1 level was dramatically increased after being infected by MSCV-EVI1 plasmid.Forced expression of EVI1 in Uocm1 significantly downregulated miR-9 by inducing hypermethylation of miR-9 promoter.Relative expression level of miR-9 was lower in EVI1 overexpressed group(0.004±0.000)than that of the control group(0.006±0.001)(t=4.09, P<0.05). When EVI1 was overexpressed in Uocm1, the rate of G0/G1 cells decreased markedly(P<0.05), while rates of S phage and G2 phage increased significantly(all P<0.05). Seven days after 500 cells plated in semi-solid medium, EVI1 overexpressed Uocm1 cells gave rise to more colony (122.3±7.8) than Uocm1 cells infected with vector(45.7±6.1)(t=-13.44, P<0.01).0.1 μmol/L 5-AZA recovered miR-9 expression(P<0.01)by decreasing EVI1 induced hypermethylation of miR-9 promoter.G0/G1 phase cell proportion was(48.25±2.19)% in control group, while (65.90±2.90)% in 5-AZA group(t=-6.85, P<0.05).5-AZA group formed less colony (51.00±10.01)than the control group (123.40±8.12) (t=9.59, P<0.01), which indicated that 5-AZA inhibited cell proliferation by G0/G1 cell cycle retardation in EVI1 overexpressed uocm1 cells. Conclusions EVI1 may enhance proliferation ability of myeloid leukemia cells by downregulating miR-9 through inducing hypermethylation of miR-9 promotor, which plays a crucial role in the pathogenesis of AML.5-AZA may be an effective hypomethylating agent in the therapy of EVI1 high acute myeloid leukemia. Key words: Ecotropic viral integration site 1; MicroRNA-9; Acute myeloid leukemia; Pathogenesis; 5-aza-2′-deoxycytidine