Cloning And Characterization Of A Hydroxyurea-inducible Gene

羟基脲诱导基因的克隆和表征

• 批准号: 7593474
• 负责人: GRIFFIN P. RODGERS
• 金额: $ 43.95万
• 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/7593474
• 关键词: 1-Phosphatidylinositol 3-Kinase; Accounting; Adult; Apoptosis; Biological Process; Blood; Blood Transfusion; Bone Marrow; CD34 gene; CDKN1A gene; Cell Cycle; Cells; Chimeric Proteins; Chromosomes, Human, Pair 10; Clinic; Cloning; Complex; Confidence Intervals; Cultured Cells; Cytotoxic agent; Data; Databases; Down-Regulation; Elevation; Erythrocytes; Erythroid; Erythroid Cells; Erythroid Progenitor Cells; Erythropoiesis; Erythropoietin; Exhibits; Fetal Hemoglobin; Frequencies; GTP-Binding Proteins; Gene Expression; Genes; Genetic Polymorphism; Genetic Transcription; Genotype; Globin; Green Fluorescent Proteins; Hemoglobin; Hospitalization; Incidence; Indium; K-562; K562 Cells; Knowledge; Laboratories; Liquid substance; Localized; MeSH Thesaurus; Mediating; Melanocyte stimulating hormone; MicroRNAs; Molecular; Multicenter Studies; Natural regeneration; Neonatal Screening; Newborn Infant; Nitric Oxide; Odds Ratio; Other Finding; POMC gene; Pain; Pathway interactions; Patients; Phase; Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide; Play; Point Mutation; Population; Production; Protein Dephosphorylation; Proto-Oncogene Proteins c-akt; Publishing; RNA Interference; Rate; Regulation; Reporting; Research; Ribonucleotide Reductase Inhibitor; Role; Sickle Cell Anemia; Sickle Hemoglobin; Signal Pathway; Signal Transduction Pathway; Soluble Guanylate Cyclase; System; Technology; Therapeutic; Transcriptional Activation; Up-Regulation; Variant; acute chest syndrome; base; beta Globin; calreticulin; cell growth; cohort; fetal; follow-up; gamma Globin; hydroxyurea; intracellular protein transport; killings; knock-down; mRNA Differential Displays; mRNA Expression; mortality; novel; oncoprotein p21; peroxidation; polymerization; programs; promoter; protein expression; protein transport; response

Hydroxyurea (HU) has been shown to augment the production of fetal hemoglobin (HbF) and for this reason is being used in the treatment of sickle cell anemia. It has been assumed that HU promote HbF production indirectly by perturbing the maturation of erythroid precursors. However, the molecular mechanism(s) involved in how HU regulates &#947;-globin expression remains unclear. By using a two-phase liquid erythroid culture system in conjunction with mRNA differential display, a HU-inducible small GTP-binding protein gene, designated SAR, was successfully cloned and its biological function is being investigated. We found the SARgene is localized to chromosome 10. SAR tagged with a GFP fusion protein is co-expressed with anti-calreticulin in the ER complex in K562 cell. Stable SAR expression in K562 cells increased &#947;-globin mRNA expression and resulted in macrocytosis and cells that appeared immature. SAR-mediated induction of &#947;-globin also inhibited K562 cell growth by causing arrest in G1/S, apoptosis, and delay of maturation, cellular changes consistent with the previously known effects of HU on erythroid cells. SAR also enhanced both gamma- and beta-globin transcription in primary bone marrow CD34+ cells, with a greater effect on gamma-globin than on beta-globin. SAR exhibits a tight correlation with gamma gene in stable transfected K562 cell, AC133+ cell cultured with EPO and K562 treated with HbF production inducers. Several pathways that contribute to the SAR induction of the gamma globin gene are being investigated. SAR upregulated GATA-2, p21, inhibited PI3 kinase and phosphorylated ERK. These data reveal a novel role of SAR distinct from its previously known protein trafficking function. SAR may participate in both erythroid cell growth and &#947;-globin production by regulating PI3 kinase/ERK and GATA-2/p21-dependent signal transduction pathways. The research data has been summarized and published in Blood 106:3256, 2005.. Based on the SAR unique biological function in gamma globin induction, SAR seems play a central role in fetal erythropoiesis. By using RNA interference technology, we are able to knock down SAR in different cells and examine related signal pathway which may illuminate how SAR regulates erythrocyte maturation and proliferation. . Our data revealed that SAR microRNA is able to down regulate HbF cell population 15-22%, block HU induced Hb F35-40% in K562 cells, and abolished HU mediated S phase cell arrest in HEK 293 cells (61-65%). SAR micro RNA also inhibited the effects of HU on PI3 Kinase protein expression, and AKT, ERK dephosphorylation. We suggest that SAR may participate in both erythroid cell growth and gamma-globin production by regulating PI3-kinase/ERK and GATA-2/p21 dependent signal transduction pathways. Our preliminary analysis indicates that HU inducibility is mediated at the transcriptional level, and is localized to elements in the SAR1A promoter. The aim of this study was to assess whether polymorphisms in the SAR1A gene promoter are associated with Hb F levels or HU therapeutic responses. We studied 386 sickle cell disease patients consisting of 269 adults treated with or without HU and 117 newborns with sickle cell disease identified from a newborn screening program. Twenty point mutations, including one nonsynonymous variant, were identified in SAR1A, including nine previously reported in SNP databases. A difference in genotype frequencies was observed between adults and newborns for rs2310991 in the 5UTR (odd ratio OR = 1.9, 95% confidence interval CI = 1.1-3.2, P=0.009) and +31 T>C in 5UTR (odd ratio OR 9.8 1.3-73.9; confidence interval CI 95%; P<0.001). Three previously unknown SNPs in the upstream 5UTR (-809 C>T, -502 G>T and -385 C>A) were significantly associated with the HbF response in Hb SS patients treated with HU (P<0.05). Our data suggest that the SAR1A polymorphism might contribute to the regulation of HbF expression and modulate patient responses to HU in sickle cell disease.

已证明，羟基脲（HU）可增加胎儿血红蛋白（HbF）的生成，因此，它被用于治疗镰状细胞性贫血。据推测，HU通过干扰红细胞前体的成熟间接促进HbF产生。然而，涉及HU如何调节β-珠蛋白表达的分子机制仍不清楚。利用两相液体培养系统结合mRNA差异显示技术，成功克隆了一个HU-inducible small GTP-binding protein基因，命名为SAR，并对其生物学功能进行了研究。我们发现SAR基因定位于10号染色体。GFP标记的SAR融合蛋白与抗钙网蛋白在K562细胞中共表达。 稳定的SAR表达在K562细胞中增加-珠蛋白mRNA的表达，并导致大红细胞和细胞出现不成熟。SAR介导的诱导-珠蛋白也抑制K562细胞的生长，引起停滞在G1/S期，细胞凋亡，并延迟成熟，细胞的变化与以前已知的作用胡红细胞一致。SAR还增强了γ-和β-珠蛋白在原代骨髓CD 34+细胞中的转录，对γ-珠蛋白的影响大于对β-珠蛋白的影响。在稳定转染的K562细胞、经EPO培养的AC 133+细胞和经HbF产生诱导剂处理的K562细胞中，SAR与γ基因密切相关。几个途径，有助于SAR诱导的γ珠蛋白基因正在调查。SAR上调加塔-2、p21，抑制PI 3激酶和磷酸化ERK。这些数据揭示了SAR不同于其先前已知的蛋白质运输功能的新作用。SAR可能通过调节PI 3激酶/ERK和加塔-2/p21依赖的信号转导通路参与红系细胞生长和β-珠蛋白的产生。研究数据已总结并发表在Blood 106：3256，2005中。 基于SAR在γ-珠蛋白诱导中独特的生物学功能，SAR可能在胎儿红细胞生成中起重要作用。利用RNA干扰技术，我们可以在不同的细胞中敲除SAR，并研究相关的信号通路，这可能有助于阐明SAR如何调节红细胞的成熟和增殖。.我们的数据显示，SAR microRNA能够下调HbF细胞群体15- 22%，阻断K562细胞中HU诱导的HbF 35 -40%，并消除HEK 293细胞中HU介导的S期细胞阻滞（61-65%）。 SAR microRNA还可抑制HU对PI 3激酶蛋白表达的影响，抑制AKT、ERK去磷酸化。我们认为SAR可能通过调节PI 3-激酶/ERK和加塔-2/p21依赖的信号转导通路参与红系细胞生长和γ-珠蛋白的产生。 我们的初步分析表明，HU诱导是在转录水平介导的，并定位于SAR 1A启动子中的元件。 本研究的目的是评估SAR 1A基因启动子多态性是否与Hb F水平或HU治疗反应相关。我们研究了386名镰状细胞病患者，包括269名接受或不接受HU治疗的成年人和117名新生儿，这些新生儿从新生儿筛查项目中确定患有镰状细胞病。 在SAR 1A中发现了20个点突变，包括一个非同义变异，其中包括9个以前在SNP数据库中报道的点突变。 在成人和新生儿之间观察到5 UTR中rs 2310991（比值比OR = 1.9，95%置信区间CI = 1.1-3.2，P=0.009）和5 UTR中+31 T>C（比值比OR 9.8 1.3-73.9;置信区间CI 95%; P<0.001）的基因型频率存在差异。在用HU治疗的Hb SS患者中，上游5 UTR中的三个先前未知的SNP（-809 C>T、-502 G>T和-385 C>A）与HbF反应显著相关（P<0.05）。 我们的数据表明，SAR 1A多态性可能有助于调节HbF的表达和调节患者对HU的反应在镰状细胞病。