Erythropoietin-Inducible MicroRNA-362 Contributes to Erythropoiesis Via the Suppression of Histone Deacetylase 3.

Abstract 2603

Erythropoietin (EPO) is crucial for various aspects of erythropoiesis, such as proliferation, differentiation and maturation through the regulation of downstream genes including anti-apoptotic regulator, Bcl-XL. However, there is still no answer about the detail list of the downstream genes of EPO. Therefore, we tried to identify EPO-inducible microRNAs (miRNAs) which are a novel class of regulatory molecules. MiRNAs fine-tune gene expression that is likely to be important for a wide range of cellular functions, including the hematopoietic system. We screened specific miRNAs expressed in human leukemia cell lines, UT-7 and its sublines UT-7/GM, UT-7/EPO and UT-7/TPO which are all differentiated from UT-7 cells by the cytokine stimulation. In an EPO-dependent subline, UT-7/EPO, miR-188 and miR-362 were highly expressed compared to the other sublines. An increased expression of both miR-188 and miR-362 in response to stimulation by EPO was observed in human erythroleukemia cell line TF-1 cells and a EPOR expressing FDCP2 cells. Furthermore, we investigated the expression of miR-188 and miR-362 in enhanced erythropoiesis following PHZ-induced hemolytic anemia in mice. The expression of both miRNAs was increased 3- to 6-fold in spleen cells of PHZ-treated mice. In addition, the expression of miR-188 was gradually up-regulated, whereas the expression of miR-362 was down-regulated during erythroid differentiation in the primary culture. These results showed that the expression of miR-188 and miR-362 was induced by EPO stimulation and indicated the involvement of both miRNAs in post-transcriptional regulation during erythropoiesis. To narrow down the putative mRNA targets of miR-362, we used in silico algorithms (TargetScan, Sanger MiRBase, and MiRTarget2), restricting to the erythropoietic process-related genes. According to these criteria, histone deacetylase 3 (HDAC3) emerged as a candidate. Previous study reported that HDAC3 represses the transcription factor GATA-2 that plays essential roles in hematopoietic stem and progenitor cell compartments. Transfection of miR-362 mimic molecule decreased the expression of HDAC3 compared to the negative control (NC) in UT-7 and UT-7/EPO cells. To prove that miR-362 directly recognizes the identical predicted target site in the 3' UTR of HDAC3, that target sequence was cloned into the 3′ UTR of a Renilla luciferase reporter vector to generate HDAC3-UTR-report. MiR-362 mimic molecule significantly downregulated Renilla luciferase activity, demonstrating that miR-362 represses gene expression by directly recognizing the predicted target sequence in the 3′ UTR of HDAC3. Consequently, our study shed the light on a new aspect of EPO mediated erythropiesis, suggesting that EPO-inducible miR-362 regulates the epigenetic status of erythrocyte through the suppression of HDAC3 and the activation of GATA-2.