Erythropoietin-Inducible MicroRNA-362 Contributes to Erythropoiesis Via the Suppression of Histone Deacetylase 3.
Authors: Yoshioka Yusuke , Sugiura Keiichi , Kosaka Nobuyoshi , Yamamoto Yusuke , Komatsu Norio , Miyazaki Hiroshi , Ochiya Takahiro , Kato Takashi 
Published: October 2019

Abstract
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.

Disclosures:
Miyazaki: Kyowa Hakko Kirin Co., Ltd: Employment.