IDENTIFICATION OF GENE EXPRESSION IN POLYCYTHEMIA VERA BY DIFFERENTIAL DISPLAY

通过差异显示鉴定真性红细胞增多症的基因表达

• 批准号: 6289741
• 负责人: GRIFFIN P. RODGERS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289741
• 关键词: cell population study; erythroid stem cell; erythropoiesis; erythropoietin; gene expression; genetic mapping; human genetic material tag; molecular pathology; northern blottings; nucleic acid sequence; polycythemia vera; polymerase chain reaction; tissue /cell culture; western blottings

Polycythemia vera (PV) is an acquired clonal hematopoietic disorder of stem cells characterized by erythrocytosis, granulocytosis and thrombocytosis. In PV there are normal erythropoietin (EPO)-dependent erythroblasts and a fraction of abnormal EPO-independent erythroid progenitors. The molecular targets responsible for these abnormal EPO- independent erythroid progenitors to proliferate and differentiate into mature red cells are still unclear. To identify the genes expressed differently in these two populations of erythroid cells, we used a two phase liquid erythroid culture system to culture and separate the EPO- dependent and EPO-independent erythroid cells from 12 phlebotomy-only treated PV patients. The polymerase chain reaction-based differential display method (DDRT-PCR) was used to identify mRNAs differentially expressed in these two subpopulation. Sixty-three mRNA transcripts display differential expression in EPO-dependent and independent groups. After using reverse dot blot to confirm and quantitate the intensity of these mRNA transcripts, twenty transcripts with downregulated or upregulated expression in EPO-independent erythroid cells compared with EPO-dependent group were cloned and sequenced. Eleven of these transcripts have high homology with already-known sequences, including B3 (beta-globin gene), B7 (ferritin heavy subunit mRNA), A3 ( Nrf1, NF-E2-related transcription factor), B1 (human nuc2 homologue), D1(human translationally controlled protein), and are downregulated in EPO-independent erythroid cells, with 26, 19, 18, 11, 7 fold lower expression in EPO-independent than in EPO-dependent groups, respectively. It has been reported that the CNC-basic leucine family member Nrf1 (A3) plays an essential role during fetal liver hematopoiesis; mice homozygous for the Nrf1 mutation suffer from anemia as a result of abnormal fetal liver hematopoiesis. The human homologue of yeast nuc2 (B1) has been reported to interacts with the Rb protein in a specific manner. The expression of A3, B1 and D1 in 12 PV patients was confirmed by Northern blot. The role of A3, B1 and D1 in PV is under investigation. Nine of twenty transcripts have no homology with known sequences. Three of nine unknown transcripts were upregulated in the EPO-independent group. By Northern blot analysis, their expression and size were confirmed. Their full cDNA sequence is being obtained by using a human cDNA library. Investigation of both downregulated and upregulated cDNA transcripts in EPO-independent erythroid cells may provide insights into the pathogenesis of and possible therapeutic targets in PV.

真性红细胞增多症（PV）是一种以红细胞增多、粒细胞增多和血小板增多为特征的获得性干细胞克隆性造血系统疾病。在PV中，存在正常的促红细胞生成素（EPO）依赖性成红细胞和一部分异常的EPO非依赖性红系祖细胞。负责这些异常EPO非依赖性红系祖细胞增殖和分化成成熟红细胞的分子靶点仍不清楚。为了鉴定在这两个红系细胞群体中不同表达的基因，我们使用两相液体红系细胞培养系统培养并分离来自12名仅放血治疗的PV患者的EPO依赖性和EPO非依赖性红系细胞。采用基于聚合酶链反应的差异显示技术（DDRT-PCR）对两个亚群中差异表达的mRNA进行鉴定。63个mRNA转录物在EPO依赖组和非依赖组中显示差异表达。经反向斑点杂交证实并定量这些mRNA转录本的强度后，克隆了20个在EPO非依赖性红系细胞中表达下调或上调的转录本，并进行了测序。这些转录本中有11个与已知序列具有高度同源性，包括B3（β-珠蛋白基因），B7（铁蛋白重亚基mRNA），A3（Nrf 1，NF-E2相关转录因子），B1（人nuc 2同源物），D1（人免疫控制蛋白），并且在EPO非依赖性红系细胞中下调，其中26，19，18，11，EPO非依赖组的表达分别比EPO依赖组低7倍。据报道，CNC-碱性亮氨酸家族成员Nrf 1（A3）在胎肝造血过程中发挥重要作用; Nrf 1突变纯合子小鼠因胎肝造血异常而患有贫血。据报道，酵母nuc 2（B1）的人类同源物以特异性方式与Rb蛋白相互作用。北方杂交证实12例PV患者中有A3、B1和D1的表达。A3、B1和D1在PV中的作用正在研究中。20个转录本中有9个与已知序列没有同源性。在EPO非依赖组中，9个未知转录物中有3个上调。通过北方印迹分析，证实了它们的表达和大小。通过使用人cDNA文库获得它们的全长cDNA序列。研究EPO非依赖性红系细胞中下调和上调的cDNA转录物可能为PV的发病机制和可能的治疗靶点提供见解。