Analysis Of Erythroid Differentiation-associated MicroRNAs And Primary Study On Their Function In Hematopoiesis

Erythropoiesis is a process by which pluripotent hematopoietic stem cells give rise to erythrocytes undergoing a series of proliferation and differentiation decision stages.It is a complicated and elaborate process controlled by opening and closing of related genes in proper order or increasing and decreasing expression of related genes.MicroRNAs(miRNAs) are a novel class of conserved 21-23 nucleotides long RNAs which can negatively regulate gene expression at post-trancriptional level by mRNA degradation or translation repression.So far,more than 500 human miRNAs have been reported and by estimation about 30%human genes were controlled by miRNAs.K562 is a cell line coming from chronic myeloid leukemia,which is situated in the common progenitor stage of megakaryocytic and erythroid lineages of the hematopoietic stem cell differentiation and its normally following differentiation is blockaded.However, K562 cells can be induced to erythroid differentiation and megakaryotic differentiation by hemin and Phorbol-12-myristate13-acetate(PMA) respectively.To get more information on gene regulation during erythroid differentiation of K562 cells,we analysis 435 human miRNAs expression in K562 cells and hemin-induced K562 cells by microarray.We identified 31 miRNAs expressed in un-induced K562 cell and 8 of them(miR-92,miR-17-3p,miR-19a, miR-19b;miR-126,miR-18a,miR-17-5p and miR-20b) with relative high expression.We compared the miRNA expression of hemin-treated cells with untreated cells using Significance Analysis of Microarrays and detected 15 miRNAs with different expression. Among them seven miRNAs,miR-185,miR-422a,miR-130b,miR-451,miR-20b, miR-126 and miR-20a appeared up-regulation and eight miRNAs,miR-25,miR-210, miR-223,miR-27a,miR-103,miR-18b,miR-10a and miR-130a exerted down-regulation in the hemin-induced K562 cells.We then analyzed expression of theses miRNAs by Northern blot and confirmed the increase of miR-126 and miR-451 whereas the decrease of miR-103,miR-130a,miR-210 and miR-18b during heroin-induced K562 erythroid differentiation.Northern blot analysis indicated that miR-146b increased obviously after hemin-induced erythriod differentiation and PMA induced megakaryotic differentiation. The expression of miR-126,miR-451,miR-103,miR-130a,miR-210,miR-18b and miR-146b in 10 leukemia cell lines was also examined and most of them exhibited enrichment in erythroleukemia cell lines(K562 or/and HEL).The expression of theses miRNAs indicated they may have important roles in erythroid differentiation.We examined the expression of miR-126,miR-451,miR-103,miR-130a,miR-210, miR-18b and miR-146b during the erythroid differentiation of purified cord blood CD34+ progenitors in a liquid culture system by real-time PCR.An obviously increased miR-126, miR-451 and miR-146b expression whereas a significantly decreased expression of miR-103,miR-130a,miR-210 and miR-18b were detected during erythroid differentiation of CD34+cells.The results were in coincidence with hemin-induced K562 cell differentiation.These data indicated that miR-451,miR-126,miR-146b,miR-103, miR-130a,miR-210 and miR-18b may take part in normal erythropoiesis.The down-regulation of miR-103 in the process of erythroid differentiation suggested that miR-103 may participate in hematopoiesis.In accordance with our hypothesis,K562 cells transfected with miR-103 precursor showed a marked decrease of the benzide positive cells compared with cells transfected with the control oligonucleotides and untransfected K562 cells with miR-103 over-expression inhibits hemin-induced K562 erythroid differentiation.Contrary to miR-103,the expression of miR-146b increased in erythroid differentiation.Temporal over-expression of miR-146 could promote hemin-induced K562 cell erythroid differentiation and PMA-induced K562 cell megakaryocytic differentiation. Stable transfectant with enforced expression of miR-146b in K562 was constructed and its character of erythroid differentiation was examinated.Compared with K562 cells,benzide positive cells increased greatly both in heroin treated and untreated cells.MiRNAs exert function by down-regulate protein expression of their target genes. Here we analyzed the targets of miR-103 and miR-126.By using three target prediction algorithms,10 predicted targets that were reported related to cell differentiation,were selected for further examination.To demonstrate a direct interaction between the 3'-UTR of probable targets with miR-103,we inserted the 3'-UTR region of each mRNA predicted to interact with miR-103 into a luciferase vector pRL-TK.More than 50%reduction of luciferase level was observed in Hela cells co-transfected with pre-miR-103 together with forkhead box J2(FOXJ2)-UTR.In contrast,no changes in other reporter plasmid expression were observed in the presence of oligonucleotide pre-miR-103.To confirm whether over-expression of miR-103 in vivo suppresses FOXJ2 protein expression,we measured FOXJ2 protein by Western blot and it's mRNA by real-time PCR.As expected, FOXJ2 protein reduced greatly in Hela cells transfected with pre-miR-103 compared with the Hela cells transfected with the control dsRNA,while its mRNA level had no significant change.Additionally a significantly decreased FOXJ2 expression was also observed in K562 cells transfected with pre-miR-103 compared with the K562 cells transfected with the control dsRNA.The similar method was used to demonstrate PTPN9 was the target of miR-126.Five putative targets including PTPN9(protein tyrosine phosphatase,non-receptor type 9) and CRK(v-crk sarcoma virus CT10 oncogene homolog) mRNA were selected out from all probable targets predicted by the three target prediction algorithms for further study. Luciferase reporter assays were carried out to confirm directed interaction of miR-126 and the 5 putative targets.An obviously decreased luciferase activity was observed in Hela transfected with luciferase taking pRL-TK-PTPN9-UTR and pRL-TK-CRK-UTR.Finaly, by Western blot analysis with anti-PTPN9 antibody,a significantly reduced PTPN9 protein was detected in the cells with miR-126 over-expression.FOXJ2 is a member of forkhead family of transcription factors that have been proven important in processes such as proliferation,differentiation and survival for embryonic development,hematopoietic T cell differentiation and neural systems.Inhibiting erythroid differentiation of K562 by enforced expression of miR-103 may exert by downregulation of FOXJ2.PTPN9 is a member of the protein tyrosine phosphatase(PTP) family.Several PTP family members are known to be signaling molecules that regulate a variety of cellular processes including cell growth,differentiation,mitotic cycle and oncogenic transformation.PTPN9 was found important to the development of erythroid progenitor cells.The higher PTPN9 activity may have higher capability to proliferate and survive, thus producing more erythrocytes upon maturation and producing the erythroid hyperplasia in PV(polythemia vera) patients.Here we confirmed the modulation of PTPN9 by miR-126,putting forward a new regulating mechanism for erythroipoiesis.We observed that the PTPN9 mRNA in K562 cells up-regulated intensely after Hemin induction for 24 hours,whereas its protein level had no visible change.Since the increased miR-126 expression during hemin-induced K562 erythroid differentiation was detected,the uncorrelated PTPN9 mRNA and protein change pattern may result from down-regulation of PTPN9 protein expression by increased miR-126.Increasing of miR-126 in the process of hemin-induced K562 erythroid differentiation may prevent intense and rambunctious augment of PTPN9,which help to hold out the balance crucial for hematopoiesis.It is now well accepted that miRNAs co-operated to regulate gene expression via translational repression or mRNA cleavage.MiRNAs function at post-transcriptional level makes up efficient complementarity to transcription regulation of erythroid differentiation control.Here,we explored miRNA expression patterns in K562 cells,identified a subset of miRNAs varied in erythroid differentiation,analyzed the function of miR-103 and miR-146b in erythroid and megakaryocytic differentiation,found the targets of miR-103 and miR-126.Our data may offer new insight into the molecular mechanism in human erythropoiesis and erythroleukemia tumorgenesis.Our results supply the significant data and important clues for the detailed mechanisms and regulation network of erythroid differentiation and possibly provide potential clues for studying and curing the diseases related to hematopoietic differentiation.