MiR34a Contributes to Defective Erythropoiesis in RPS19 Insufficient Hematopoietic Stem Cells

Abstract 2404

Diamond-Blackfan Anemia (DBA) is characterized by defective erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a defect in the RPS19 gene, which encodes a ribosomal protein on the 40S ribosomal subunit. Previous studies in zebrafish and mice have shown that knockdown of RPS19 is correlated with the upregulation of p53, a tumor suppressor gene that regulates cell cycle and apoptosis. However, the molecular pathways that link ribosome dysfunction and defective erythropoiesis remain unknown.

Among the targets of p53 is microRNA34a (miR34a). Overexpression of miR34a in human myeloid leukemia K562 cells inhibits cell proliferation, leads to cell cycle arrest at the G1 stage, and promotes differentiation toward the megakaryocytic lineage in a p53-independent manner. MiR34a downregulates numerous proteins, including c-Myb, which is essential for normal erythropoiesis and has been found to be expressed at lower levels in bone marrow cells from DBA patients. We hypothesize that miR34a induction downstream of p53 activation in RPS19 insufficient normal hematopoietic progenitor cells contributes to defective erythropoiesis observed in DBA patients.

To study the effects of RPS19 insufficiency and downstream signaling pathways, we transduced primary CD34+ fetal liver cells with two different shRNA constructs against RPS19. Knockdown was confirmed by Western blot analysis and by qRT-PCR (73%, p< 0.0001 for shRNA1, and 87%, p< 0.0001 for shRNA2, respectively), compared with a scrambled control shRNA. Cells were sorted for a GFP marker 72 hours post-transduction and either collected for downstream analysis or propagated further in methylcellulose.

Our results demonstrated that knockdown of RPS19 in primary human CD34+ fetal liver cells leads to an increase in p53 protein by Western blot analysis, and upregulation of miR34a mRNA (3-fold, p<0.01). This upregulation correlates with downregulation of miR34a target genes including c-Myb (p<0.01) and c-Myc (p<0.01). Induction of miR34a and downregulation of c-Myb and c-Myc, occurs through a p53-dependent pathway since the expression of those genes was not altered in p53-null K562 cells transduced with RPS19 shRNA lentivirus. To correlate molecular pathways with the differentiation potential of RPS19 insufficient CD34+ fetal liver cells, methylcellulose colony assays were performed. Total colony formation was reduced by 87% (p<0.01) in cells treated with RPS19 shRNA compared to scrambled control shRNA. Erythroid colony formation in methylcellulose containing IL-3, SCF, GM-CSF, and EPO, decreased by 90% (p<0.01) in shRNA transduced cells compared to control shRNA. FACS analysis for erythroid markers CD71 and Glycophorin A showed a similar pattern, with the CD71+GlyA+ population representing 2.6% of RPS19 shRNA transduced cells, compared to 9.9% in cells transduced with the scrambled control. Preliminary data with lymphoblastoid cell lines (LCLs) from DBA patients with RPS19 insufficiency demonstrate 2-fold upregulation of miR34a. Therefore, we conclude that RPS19 insufficiency in human CD34+ fetal liver cells leads to p53-dependent upregulation of miR34a, down-regulation of miR34a target genes c-Myc and c-Myb, and defects in erythroid differentiation.