MiR-101 Down Regulates Neuromedin U Expression During Human Erythropoiesis and Negatively Regulates Erythroid Expansion,
Authors: Jackson Emily , Brito Giselle , Li Ningkun , Carroll Martin , Shetzline Susan E. 
Published: October 2019

Abstract
Abstract 3160
Recently, we reported that the neuropeptide, neuromedin U (NmU), functions as a novel extracellular cofactor with erythropoietin (EPO) to promote the expansion of early human erythroblasts. Because the expression of NmU is important during the early stages of erythropoiesis, we aimed to understand its temporal regulation during erythroid development. Although we have demonstrated that NmU is a target of the erythroid transcriptional regulator, c-Myb, our understanding of NmU regulation is incomplete. We hypothesized that microRNA (miRNA) molecules function to regulate NmU expression at the post-transcription level during erythropoiesis. Upon sequence analysis of the 3'-UTR of NmU using microCosm in the miRBase Targets database, 20 different miRNA molecules were predicted to interact with NmU's 3'-UTR. Among the 20 different miRNA molecules predicted to interact with NmU's 3'UTR, miR-101 was of interest, because in an independent study, its expression was elevated as measured by microarray analyses from primary human CD34+ cells cultured under erythroid inducing conditions. To determine the ability of miR-101 to directly interact with the 3'UTR of NmU, we used luciferase reporter assays. In a dose-dependent manner, miR-101 directly interacted with NmU's 3'-UTR. Also, 24-hours post-nucleofection of miR-101 into K562 cells, a hematopoietic cell line, the expression of NmU was decreased compared to control. Over-expression of miR-101 in primary human CD34+ cells decreased the growth of colony-forming unit-erythroid (CFU-E) ∼50% compared to control cells. In the presence of exogenously added NmU peptide, CFU-E growth from CD34+ cells over-expressing miR-101 was rescued to the level observed with control miRNA treated cells. To further determine the relationship between NmU, EPO, and miR-101, we cultured primary human CD34+ cells using a 2-phase liquid culture condition to induce erythroid development. During the first phase (days 0–6), the cells were cultured with IL-3, IL-6, and stem cell factor (SCF). The second phase of the erythroid inducing culture conditions began on day 6 when EPO was added to the culture. Erythroid differentiation was monitored using flow cytometry and fluorescent conjugated antibodies against CD34, transferrin receptor (CD71), and glycophorin A (GlyA). In parallel, primary cells were collected at regular intervals during culture to measure the expression of NmU mRNA and miR-101 by real time PCR (RT-PCR). Under our erythroid inducing culture conditions, NmU expression peaked between days 4 and 6 (before adding EPO) and between days 10 to 12. Also, between days 10 to 12 of culture in erythroid inducing conditions, we observed a dramatic increase in cell proliferation. Between days 13 to 15, cell proliferation reached a plateau, and the expression of miR-101 peaked. Erythroid progenitors purified from cord blood mononuclear cells by cell sorting revealed that NmU expression peaked in CD34-, CD71+, GlyA- (ERY2) cells, which is in good agreement with an independent microarray study, and miR-101 expression was not detected. By contrast, in CD34-, CD71lo, GlyA+ (ERY4) cells, miR-101 expression peaked while NmU expression decreased to the level observed in CD34-, CD71-, GlyA- cells. Combined, these data identify NmU as a novel miR-101 target and indicate that miR-101 regulates the temporal expression of NmU during the later stages of erythropoiesis. We hypothesize that the miR-101/NmU axis is a critical modulator of erythroid cell expansion that augments the effects of erythropoietin.

Disclosures:
Carroll: Glaxo Smith Kline, Inc.: Research Funding; Sanofi Aventis Corporation: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.