Regulation of miR-210 Generation in Response to Hypoxia in Erythrocytic Cells.

EPO-EPOR-mediated intracellular signaling activates JAK/STAT, ras/raf/MAP kinase and PI3 kinase/Akt cascades to promote cell survival, proliferation and differentiation. Alternatively, recent studies suggest that microRNAs (miRNAs) are a novel class of small noncoding RNAs that regulate gene expression at the post-transcriptional level and play a critical role in many important biological processes. Although recent reports showed the importance of miRNA functions in hematopoietic cells, the effect of EPO on miRNA expression and the role of miRNA in modulating EPO-mediated cell proliferation and differentiation are not reported yet. In this study, we investigate the function of miRNAs in factor-dependent human cell lines, EPO-dependent UT-7/EPO and GM-CSF-dependent UT-7/GM. We employed a human miRNA microarray to detect 470 miRNAs in order to evaluate the expression patterns of miRNA between UT-7/EPO and UT-7/GM. The expression of miR-210 in UT-7/EPO was 30-fold higher than that in UT-7/GM. The EPO-dependent cell lines TF-1 showed substantially increased levels of miR-210 by stimulation with EPO, compared to expression in TF-1 cells maintained by GM-CSF. Recently, miR-210 was identified as a group of miRNAs that were induced in a hypoxic environment in colon and breast cancer cell lines (Kulshreshtha et al., MCB. 2007). Enforced expression of miR-210 decrease proapoptotic signaling in a hypoxic environment in breast cancer cell line. To investigate the regulation of miR-210 in hematopoietic cells under hypoxic condition, expression level of miR-210 was determined and compared between hypoxia (1% O2) or normoxia (21% O2) in UT-7 cell lines. The expression of miR-210 increased 5- to 80-fold in UT-7, UT-7/GM and TF-1 under hypoxic condition. UT-7/EPO expressed high level of miR-210 in normoxia and maintained the same level in hypoxia. To examine whether the transcription of miR-210 primary transcripts (pri-miR-210) is regulated by hypoxia signaling, pri-miR-210 was detected by RT-PCR in hypoxia- or normoxia-treated cells. Higher expression of pri-miR-210 was detected in hypoxia-treated cells. Through sequence analysis, several putative hypoxia response elements that are highly conserved among different species were identified in the 5′-upstream region of pri-miR-210. These observations indicated that the transcription of pri-miR-210 was at least in part controlled by HIF-1 α protein. It is well known that hypoxic stress strongly enhances erythropoiesis, but the effect of hypoxia on erythroid progenitors has not been examined precisely. HIF mediates short-term adaptive responses to hypoxia, and several glucose transporters and glycolytic enzymes are transcriptionally regulated by HIF, as well as genes involved in cell growth and survival. In this view, we hypothesize that the physiological function of miR-210 is erythroid cell survival in the hypoxic stress. In silico analysis revealed candidate targets that are part of the apoptotic machinery and glucose metabolism. Therefore, we examined whether miR-210 is expressed in primary erythrocytic cells. These findings suggest a novel pathway that miR-210 contribute to the erythroid cell survival in hypoxic condition.