| Hematopoiesis is one of the most important links in organism development and lifemaintenance. Regulation of hematopoiesis consists of two integral aspects: (1) theself-renewal of hematopoietic stem cells (HSCs)—how pluripotential stem cellsreplicate themselves; (2) the differentiation of hematopoietic stem/progenitor cells—howstem/progenitor cells undergo progressive differentiation decision of at least eightdistinct lineage potential and develop into mature, terminally differentiated cells.Erythropoiesis is a process by which pluripotent hematopoietic stem cells give riseto erythrocytes undergoing a series of differentiation decision and proliferation stages byopening and closing of related genes in proper order or increasing and decreasingexpression of related genes. K562 is an erythroleukemia cell line, which is situated in thecommon progenitor stage of megakaryocytic and erythroid lineages of the hematopoieticstem cell differentiation and its normally following differentiation is blockaded. However,K562 cells can be induced to erythroid differentiation by hemin. To get more informationon gene regulation during erythroid differentiation of K562 cells, we applied modifieddifferential display reverse transcription polymerase chain reaction (DDRT-PCR) methodto identify genes showing differential expression in uninduced and hemin-induced K562cells. We totally obtained 357 differential ESTs. We selected 314 cDNA fragments forre-amplifying, cloning, sequencing and analyzing with bioinformatics methods. Amongthem, 201 were upregulated and 85 downregulated; 237 differential ESTs were found tohave more than 95% homology to known GenBank sequences and 45 represented cDNA sequences with only dbEST database matches and 4 ESTs have no any database matches.These known genes are involved in cell signaling transductors, factors associated cancerand apoptosis, factors associated differentiation, chromatin structure proteins, enzymesassociated redox state, some unknown function cDNAs and unknown genes. At the sametime, we also obtained 21 differential miRNAs with differential expression s inuninduced and hemin-induced K562 cells by miRNA-Microarry analysis. Our resultsprovide vital clues for identifying genes and regulation network related to erythroiddifferentiationMicroRNAs (miRNAs) are a novel class of conserved 21-23 nucleotides long RNAswhich can negatively regulate gene expression at post-trancriptional level by mRNAdegradation and translation repression. So far, one thousand of miRNAs have been foundin animals and plants. They may have diverse biological functions in developmental andphysiological processes, but only a handful of miRNAs have been carefully studied.Recently, mir-223 was reported to be specifically expressed in bone marrow of mice.Here, to study the function of mir-223 in human hematopoiesis, we firstly analyzed theexpression of mir-223 in human normal tissues, and found that its expression was higherin lymphonode, liver and bone marrow, whixh is different to published mouse data byBartel et al. Then we isolated 5 normal hematopoietic lineage cells (T cells, Bcells,granulocytes, monocytes and erythrocytes) from cord blood by density gradientcentrifugation and magnetic cells sorting and detected the expression of mir-223 in them.mir-223 expression was low in T cells and erythrocytes, moderate in B cells and high inmyeloid cells. Subsequently we also detected mir-223 expression in 9 human leukemiacell lines and found that its expression was high not only in HL-60 and THP-1 derivedfrom myeloid cells but also in 2 erythroleukemia cell lines—K562 and HEL. However,we failed to detect its signal in two mouse erythroleukemia cell lines—MEL585 andGM979. These cases indicated that miRNA may posses more biological function inhuman than mouse in diverse developmental and physiological processes. To studypossible functions of mir-223, we examined if expression inhibiting or overexpressing of mature mir-223 could affect growth and differentiation of K562 and HEL cells. Resultsshowed that inhibiting or overexpressing mature mir-223 exogenously did not influenceproliferation and apoptosis of the cells, but inhibiting mature mir-223 exogenously couldincrease the ratio of cells that express globins in hemin-induced HEL and K562 cells.This case implied that human mir-223 meight possess inhibition function for erythroiddifferentiation of K562 and HEL cells. Further studies should be performed todemonstrate this observation.Recently, following the discovery of miRNA gene clusters, some groups indicatedthat miRNAs might work in combination to accomplish their function throughout manybiological processes. In this study, we first analyzed the clustering properties of allregisted 326 human miRNA genes and found that 148 miRNA genes are organized in atotal of 51 clusters. Subsequent promoters analysis demonstrated our results. Alignmentof the miRNA sequences in different clusters revealed a significant number of miRNAparalogs among the clusters, implying an evolution process targeting the potentiallyconserved roles of these molecules. Then we performed northern blot analysis toexamine expression profiling of all clustered miRNAs in several human leukemia celllines. Consistent expression of the miRNAs in a single cluster was revealed in 39 clusters,while inconsistent expression of members in a single cluster was detected in the other 12clusters. Meanwhile, we identified several hematopoietic lineage-specific or -enrichedmiRNA clusters (e.g., the mir-29c, mir-302, mir-98, mir-29a and let-7a-1 clusters) andindividual miRNAs (e.g., mir-181 c, mir-181 d, mir-191 and mir-136). These findings maysuggest vital roles of these miRNA clusters or miRNAs in human hematopoiesis andoncogenesis, and provide clues for better understanding the function and mechanism ofmiRNAs in various biological processes.Our results provide the significant data and important clues for the detailedmechanisms and regulation network of hematopoiesis and especial erythroiddifferentiation, and possibly provide potential clues for studying and curing the diseasesrelated to hematopoietic differentiation.
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