红系分化相关miRNA的鉴别与功能研究
摘要
红细胞生成经历了骨髓多能干细胞向红系的分化决定以及红系祖细胞的继续分化和发育成熟。这一过程涉及多个基因有序开启和关闭的复杂而又精密的调控。
小RNA(microRNA,miRNA)是一类长度约为21-23个核苷酸的重要调节分子,它可以通过靶mRNA剪切或翻译抑制的方式在转录后水平抑制蛋白编码基因的表达。迄今共发现了500多种人类miRNAs,估计约有30%的人类基因被miRNAs调节。
K562细胞系是红白血病细胞系,处于多能髓样祖细胞阶段而停止继续分化。在氯高铁血红素(hemin)诱导下可继续红系分化过程,在佛波酯(PMA)的诱导下可以向巨核系分化。我们应用小RNA芯片分析了hemin诱导K562细胞红系分化前后miRNA表达的变化,发现31个在K562细胞中表达明显的miRNAs,其中8个,miR-92、miR-17-3p、miR-19a、miR-19b、miR-126、miR-18a、miR-17-5p和miR-20b呈现高表达。
比较hemin诱导K562细胞红系分化前后miRNA的表达,发现15个表达变化的miRNAs。其中上调的有7个miRNAs,miR-185、miR-422a、miR-130b、miR-451、miR-20b、miR-126和miR-20a;8个下调的miRNAs,miR-25、miR-210、miR-223、miR-27a、miR-103、miR-18b、miR-10a和miR-130a。用Northern blot验证芯片结果,显示miR-103、miR-130a、miR-18b和miR-210在红系分化过程中降低;miR-126和miR-451在这一过程中升高,与芯片结果一致;miR-146b在hemin、PMA诱导的K562细胞红系和巨核系分化过程中都明显升高。提示它们可能在红系分化过程中发挥重要作用。
用Northern blot方法检测了miR-103、miR-130a、miR-18b、miR-210、miR-126、miR-451和miR-146b在10种白血病或淋巴瘤来源的细胞系的表达,发现除miR-103外,其余的miRNAs在红白血病细胞系(K562或HEL)表现出一定程度的富集。
进一步用实时定量PCR(Real-time PCR)方法检测了这些miRNAs在脐血来源的CD34+干/祖细胞体外红系诱导培养过程中的表达变化,发现与K562红系分化过程中的变化趋势一致,即miR-126、miR-451和miR-146b在红系分化过程中明显升高;miR-103、miR-130a、miR-210和miR-18b在这一过程中明显降低。进一步提示这些miRNAs可能在红系分化中发挥重要调控作用。
我们进一步研究了miR-103和miR-146b在红系分化过程中的功能。用寡核苷酸miR-103前体瞬时转染K562细胞,观察增加miR-103表达对红系分化的影响。结果显示过表达miR-103的K562细胞与对照细胞相比,在hemin诱导后不同时间点联苯胺染色阳性细胞比率都明显降低。说明过表达miR-103能够在一定程度上抑制hemin诱导的K562细胞红系分化。
为了研究miR-146b在红系、巨核系分化中可能的作用,我们构建了过表达miR-146b的重组质粒pcDNA-146b。瞬时转染K562细胞后,观察到过表达miR-146b的K562细胞表现出较高水平的红系和巨核系分化能力。在过表达miR-146b的pcDNA-146b/K562稳定株中得出与瞬时转染相同的结果,无论在诱导前还是诱导后不同时间点联苯胺染色阳性细胞数量都明显高于K562细胞。
进一步试验了miR-103和miR-126在红系分化中的作用机制。首先通过软件预测筛选到10个miR-103可能的靶基因。将这些候选靶基因3′UTR区可能与miRNA作用的位点分别克隆到萤光报告基因下游,与miR-103前体共同转染Hela细胞后,观察过表达miR-103对萤光素酶表达的影响。结果发现过表达miR-103能够使克隆有叉头蛋白家族转录因子J2(forkhead box J2,FOXJ2)3′UTR区的报告质粒萤光素酶的表达量降低50%。用Real-time PCR和Westen blot的方法检测过表达miR-103的Hela细胞和K562细胞中FOXJ2 mRNA和蛋白的表达变化,发现过表达miR-103后,在不改变FOXJ2 mRNA的情况下能够明显降低FOXJ2蛋白表达,证实了FOXJ2是miR-103的靶基因。
利用相似的方法鉴别了miR-126的靶基因。首先用预测软件筛选到5个与红系分化有关的可能的靶基因,进一步用萤光报告基因方法观察到携带CRK(v-crk sarcomavirus CT10 oncogene homolog)和蛋白酪氨酸磷酸酶非受体亚单位9(protein tyrosinephosphatase,non-receptor type 9,PTPN9)3′UTR区的报告质粒萤光素酶的表达量在过表达miR-126时明显降低。最后用Westen blot方法证实了PTPN9蛋白的表达也在miR-126升高后明显降低。
FOXJ2是具有叉头结构转录因子的一种,已有试验证实这类因子参与了多种细胞的增殖与分化过程。推测miR-103对红系分化的作用可能是通过抑制FOXJ2表达实现的。
PTPN9是蛋白酪氨酸家族成员,已有报道这一家族在细胞生长、分化、有丝分裂和癌基因的转化等过程中具有重要作用。PTPN9的表达对于维持红细胞的增殖能力和活力方面具有重要的作用,但是过高水平的PTPN9能够使得大量的未成熟的红细胞聚集,进一步导致红细胞增多症(polythemia vera PV)。我们发现hemin诱导24小时后PTPN9 mRNA已明显升高,而它的蛋白表达几乎没有太大的变化。在红系分化过程中表达增加的miR-126可能作用于PTPN9 mRNA,防止PTPN9蛋白无限制地高水平表达。
miRNA在红系分化过程中的功能探讨为红系分化机制的研究增添了新的调控层次。我们分析了K562细胞hemin诱导前后miRNAs的表达变化,得到了一系列在K562细胞中高表达和红系诱导后变化的miRNAs;研究了miR-103在红系分化和miR-146b在红系、巨核系分化中功能作用;确定了miR-103和miR-126的靶基因。这些结果为红系分化详细机制和调控网络的揭示提供了有意义的资料和重要线索,并可能对造血细胞分化相关疾病的研究和治疗提供潜在的线索。
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.
小RNA(microRNA,miRNA)是一类长度约为21-23个核苷酸的重要调节分子,它可以通过靶mRNA剪切或翻译抑制的方式在转录后水平抑制蛋白编码基因的表达。迄今共发现了500多种人类miRNAs,估计约有30%的人类基因被miRNAs调节。
K562细胞系是红白血病细胞系,处于多能髓样祖细胞阶段而停止继续分化。在氯高铁血红素(hemin)诱导下可继续红系分化过程,在佛波酯(PMA)的诱导下可以向巨核系分化。我们应用小RNA芯片分析了hemin诱导K562细胞红系分化前后miRNA表达的变化,发现31个在K562细胞中表达明显的miRNAs,其中8个,miR-92、miR-17-3p、miR-19a、miR-19b、miR-126、miR-18a、miR-17-5p和miR-20b呈现高表达。
比较hemin诱导K562细胞红系分化前后miRNA的表达,发现15个表达变化的miRNAs。其中上调的有7个miRNAs,miR-185、miR-422a、miR-130b、miR-451、miR-20b、miR-126和miR-20a;8个下调的miRNAs,miR-25、miR-210、miR-223、miR-27a、miR-103、miR-18b、miR-10a和miR-130a。用Northern blot验证芯片结果,显示miR-103、miR-130a、miR-18b和miR-210在红系分化过程中降低;miR-126和miR-451在这一过程中升高,与芯片结果一致;miR-146b在hemin、PMA诱导的K562细胞红系和巨核系分化过程中都明显升高。提示它们可能在红系分化过程中发挥重要作用。
用Northern blot方法检测了miR-103、miR-130a、miR-18b、miR-210、miR-126、miR-451和miR-146b在10种白血病或淋巴瘤来源的细胞系的表达,发现除miR-103外,其余的miRNAs在红白血病细胞系(K562或HEL)表现出一定程度的富集。
进一步用实时定量PCR(Real-time PCR)方法检测了这些miRNAs在脐血来源的CD34+干/祖细胞体外红系诱导培养过程中的表达变化,发现与K562红系分化过程中的变化趋势一致,即miR-126、miR-451和miR-146b在红系分化过程中明显升高;miR-103、miR-130a、miR-210和miR-18b在这一过程中明显降低。进一步提示这些miRNAs可能在红系分化中发挥重要调控作用。
我们进一步研究了miR-103和miR-146b在红系分化过程中的功能。用寡核苷酸miR-103前体瞬时转染K562细胞,观察增加miR-103表达对红系分化的影响。结果显示过表达miR-103的K562细胞与对照细胞相比,在hemin诱导后不同时间点联苯胺染色阳性细胞比率都明显降低。说明过表达miR-103能够在一定程度上抑制hemin诱导的K562细胞红系分化。
为了研究miR-146b在红系、巨核系分化中可能的作用,我们构建了过表达miR-146b的重组质粒pcDNA-146b。瞬时转染K562细胞后,观察到过表达miR-146b的K562细胞表现出较高水平的红系和巨核系分化能力。在过表达miR-146b的pcDNA-146b/K562稳定株中得出与瞬时转染相同的结果,无论在诱导前还是诱导后不同时间点联苯胺染色阳性细胞数量都明显高于K562细胞。
进一步试验了miR-103和miR-126在红系分化中的作用机制。首先通过软件预测筛选到10个miR-103可能的靶基因。将这些候选靶基因3′UTR区可能与miRNA作用的位点分别克隆到萤光报告基因下游,与miR-103前体共同转染Hela细胞后,观察过表达miR-103对萤光素酶表达的影响。结果发现过表达miR-103能够使克隆有叉头蛋白家族转录因子J2(forkhead box J2,FOXJ2)3′UTR区的报告质粒萤光素酶的表达量降低50%。用Real-time PCR和Westen blot的方法检测过表达miR-103的Hela细胞和K562细胞中FOXJ2 mRNA和蛋白的表达变化,发现过表达miR-103后,在不改变FOXJ2 mRNA的情况下能够明显降低FOXJ2蛋白表达,证实了FOXJ2是miR-103的靶基因。
利用相似的方法鉴别了miR-126的靶基因。首先用预测软件筛选到5个与红系分化有关的可能的靶基因,进一步用萤光报告基因方法观察到携带CRK(v-crk sarcomavirus CT10 oncogene homolog)和蛋白酪氨酸磷酸酶非受体亚单位9(protein tyrosinephosphatase,non-receptor type 9,PTPN9)3′UTR区的报告质粒萤光素酶的表达量在过表达miR-126时明显降低。最后用Westen blot方法证实了PTPN9蛋白的表达也在miR-126升高后明显降低。
FOXJ2是具有叉头结构转录因子的一种,已有试验证实这类因子参与了多种细胞的增殖与分化过程。推测miR-103对红系分化的作用可能是通过抑制FOXJ2表达实现的。
PTPN9是蛋白酪氨酸家族成员,已有报道这一家族在细胞生长、分化、有丝分裂和癌基因的转化等过程中具有重要作用。PTPN9的表达对于维持红细胞的增殖能力和活力方面具有重要的作用,但是过高水平的PTPN9能够使得大量的未成熟的红细胞聚集,进一步导致红细胞增多症(polythemia vera PV)。我们发现hemin诱导24小时后PTPN9 mRNA已明显升高,而它的蛋白表达几乎没有太大的变化。在红系分化过程中表达增加的miR-126可能作用于PTPN9 mRNA,防止PTPN9蛋白无限制地高水平表达。
miRNA在红系分化过程中的功能探讨为红系分化机制的研究增添了新的调控层次。我们分析了K562细胞hemin诱导前后miRNAs的表达变化,得到了一系列在K562细胞中高表达和红系诱导后变化的miRNAs;研究了miR-103在红系分化和miR-146b在红系、巨核系分化中功能作用;确定了miR-103和miR-126的靶基因。这些结果为红系分化详细机制和调控网络的揭示提供了有意义的资料和重要线索,并可能对造血细胞分化相关疾病的研究和治疗提供潜在的线索。
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.
引文
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