MIR9 That Is Aberrantly Expressed in Myelodysplastic Syndrome Blocks Erythroid Differentiation Possibly Through STAT-5-Mediated Signals

Abstract 3842

microRNA (miRNA) is a class of 18–24 nucleotides small non-coding RNAs that induce mRNA degradation and negatively modulate protein translation, and plays vital roles in physiological and pathological hematopoiesis. On the other hand, expressional alterations of miRNA are postulated to result in the development of hematological malignancies including myelodysplastic syndrome. RUNX1 belongs to a member of transcription factor that is essential in the development of fetal liver hematopoiesis and is critical in the regulation of adult hematopoiesis. In myelodysplastic syndrome, dysregulated functions of RUNX1 through formation of chimeric genes or point mutations are one of the major causes of their development. We speculate that aberrant expression of miRNA that negatively regulates RUNX1 synthesis could constitute novel mechanism by which RUNX1 is abrogated. To seek for the possibility, we selected 10 microRNAs (MIR27A, MIR27B, MIR9, MIR199A, MIR18A, MIR30A, MIR30B, MIR30C, MIR30D and MIR30E) among RUNX1-related miRNAs whose seed sequences reside within the 3′ UTR of human RUNX1 mRNA as analytical objects. Investigated were 16 MDS patients (RA, 4 cases; RAEB 9, cases; RAEB-t, 2 cases; CMMoL, 1 case), 11 normal controls in our hospital and 86 MDS patients registered to MDS cell bank (RA, 23 cases; RAEB 19, cases; RAEB-t, 18 cases; CMMoL, 3 cases; MDS-derived AML, 23 cases) As a result, as for all RUNX1-related miRNAs, a tendency for higher expression levels of miRNAs in MDS samples compared to control bone marrow samples was observed. Among them, MIR9 was highly expressed in the bone marrow cells of 3 MDS cases including 1 RA case and 2 RAEB cases (approximately 19%), while barely detected in other MDS samples or controls in our institute. MIR9 was also highly expressed in 5 cases of MDS registered in cell bank including 1 RA case, 1 RAEB case, 1 RAEB-t case and 2 MDS-derived AML cases (5.8%). At first, to confirm that MIR9 actually suppresses transcription of RUNX1, we constructed reporter plasmids containing luciferase gene followed by various length of 3′ UTR of RUNX1 mRNA. As a result of reporter assay, overexpression of MIR9 decreased lucifearse activities to approximately 50 % when reporter plasmids containing miR-9 binding site in 3′ UTR of RUNX1 mRNA (2219- and 2260-) were co-transfected in 293 cells. However, when reporter plasmids without MIR9 binding site (2329-, 2411- and 3245-) were co-transfected, overexpression of MIR9 didn't suppress luciferase activities. Next, we established human hematopoietic cell line, UT-7/GM overexpressing MIR9. Evaluation by counting percentages of benzidine-positive cells, inhibition of hemoglobin synthesis by EPO treatment was observed in MIR9-overexpressing clones. Further, as a result of quantitative real-time PCR and FACS analyses, inhibition of erythroid specific gene (ALAS-E) induction and surface antigen (Glycopholin-A) expression were also observed in those clones. Finally, we analyzed protein expression levels of RUNX1 and several other intracellular signaling molecules that contribute to erythroid differentiation. There is no difference in RUNX1 protein level between mock and MIR9-overexpressing clones. Interestingly, phosphorylation of STAT-5 after EPO treatment in MIR9-overepressing clones was weaker in comparison with mock clones. All these data suggest the possibility that MIR9 functions as a negative regulator of erythroid differentiation possibly through inhibiting STAT-5 phosphorylation and that aberrant expression of MIR9 could at least partly contribute to ineffective erythropoiesis in MDS. Although, we further analyzed protein levels of JAK2 and SH-PTP2 to clarify the molecular basis of inhibition of STAT-5 phosphorylation, there was no obvious difference in expression of JAK2 or SH-PTP2 between mock and MIR9-overexpressing clones. Therefore, MIR9 must influence expression/function of unknown molecule(s) and thus may contribute to pathogenesis of MDS.