MiR-144 and MiR-451 Regulate Human Erythropoiesis By Targeting RAB14

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression by inhibiting translation and/or by degrading of target mRNAs. MiR-144 and miR-451 are expressed from a single precursor transcript, encoded by the miR-144∼451 cluster on human chromosome 17. Expression levels of both miR-144 and miR-451 are upregulated during zebrafish, mouse, and human erythroid differentiation. Several mouse and zebrafish studies have knocked down miR-451 expression and observed decreased late erythroid differentiation. In contrast, miR-144 knockdown had no or smaller effects in zebrafish or mouse erythropoiesis, and there is no study of the role of both members of this cluster in primary human erythropoiesis. Therefore, we investigated the effects of lentiviral-mediated loss-of-function of miR-144 and/or miR-451 in human erythropoiesis. We first utilized the TF1 human erythroleukemia cell line as a model, and then tested primary human CD34+ hematopoietic stem-progenitor cells (HSPCs) from normal human donors. When either TF1 or CD34+ cells are cultured with erythropoietin (EPO), the expression of the CD71 (transferrin receptor) and CD235a (glycophorin A) erythroid markers increases, and the expression of the CD34 HSPC marker decreases. In TF1 or primary CD34+ cells transduced with either a miR-144 trap or a miR-451 trap (using a lentivirus containing GFP and a series of 8 tandem complementary miR binding sequences to knock down function of either miR [Ebert, Nature Method, 2007]), as compared to control vector, erythropoiesis was decreased, as evidenced by lower numbers of erythroid (CD34^-CD71^hiCD235a^hi) cells generated during EPO-induced erythroid differentiation.

RAB14 was predicted to be a target of both miR-144 and miR-451 by the TargetScan 6.2 target prediction algorithm, with 2 predicted miR-144 binding sites and 1 predicted miR-451 binding site in its 3’UTR. RAB14 was validated to be a target of miR-451 in human non-small cell lung cancer cells [Wang, Oncogene, 2011]. RAB proteins have been reported to have important roles in vesicle trafficking, signal transduction, and receptor recycling. Recently, RAB12 was implicated in constitutive degradation of the transferrin receptor (CD71) in mouse embryonic fibroblast (MEF) cells [Matsui, Traffic, 2011], but the role of RAB14 in human erythropoiesis is unknown. We found that RAB14 protein expression decreases early during EPO-induced erythroid differentiation of TF1 cells. Then we showed that RAB14 is a direct target of miR-144, using luciferase assays and Western blots. We confirmed that shRNA-mediated RAB14 knockdown increased human erythropoiesis, as compared to control vector-transduced cells. Finally, shRNA-mediated RAB14 knockdown protected cells from miR-144/miR-451 trap-mediated erythropoietic inhibition.

In summary, miR-144 and miR-451 regulate human erythropoiesis by decreasing RAB14 expression, which in turn might have a role in regulating function of receptors involved in erythropoiesis, such as the transferrin receptor.