Cloning And Characterization Of A Hydroxyurea-inducible Gene

羟基尿素(HU)已被证明能增加胎儿血红蛋白(HBF)的产生，因此正被用于治疗镰状细胞性贫血。HU通过干扰红系前体细胞的成熟，间接促进HbF的产生。然而，胡舒立调控珠蛋白表达的分子机制(S)仍不清楚。利用两相液体红系培养系统结合mRNA差异显示技术，成功克隆了HU诱导的小GTP结合蛋白基因，命名为SAR，并对其生物学功能进行了研究。我们发现SAR基因定位在10号染色体上。在K562细胞中，标记有GFP融合蛋白的SAR与抗钙网织蛋白在内质网复合体中共表达。 K562细胞中稳定的SAR表达增加了-珠蛋白mRNA的表达，并导致巨噬细胞和看起来不成熟的细胞。SAR介导的-珠蛋白诱导也通过导致G1/S细胞停滞、细胞凋亡和成熟延迟而抑制K562细胞的生长，这些细胞变化与先前已知的HU对红系细胞的作用一致。合成孔径雷达还促进了原代骨髓CD34+细胞中的γ-和β-珠蛋白转录，对γ-珠蛋白的影响大于对β-珠蛋白的影响。稳定表达的K562细胞、经EPO培养的AC133+细胞和经HBF诱导剂处理的K562细胞的AR与γ基因密切相关。目前正在研究几种有助于伽马珠蛋白基因的合成孔径雷达诱导的途径。SAR上调GATA-2、p21，抑制PI3激酶和磷酸化ERK。这些数据揭示了SAR不同于先前已知的蛋白质运输功能的新作用。SAR可能通过调节依赖于PI3K/ERK和GATA-2/p21的信号转导通路，参与红系细胞的生长和珠蛋白的产生。研究数据已汇总并发表在《血液106：3256,2005》上。 基于SAR在丙种球蛋白诱导中的独特生物学功能，SAR似乎在胎儿红细胞生成中起着核心作用。利用RNA干扰技术，我们能够在不同的细胞中敲除SAR并检测相关的信号通路，这可能有助于阐明SAR如何调控红细胞的成熟和增殖。。结果表明，SARmicroRNA能下调K562细胞HBF细胞数15-22%，阻断HU诱导的K562细胞Hb F35-40%，阻断HU介导的HEK293细胞S期细胞停滞(61-65%)。SAR微RNA还可抑制HU对PI3Kinase蛋白表达和AKT、ERK去磷酸化的影响。我们认为，SAR可能通过调节依赖于PI3-K/ERK和GATA-2/p21的信号转导途径，参与红系细胞的生长和丙种球蛋白的产生。 我们的初步分析表明，HU的诱导作用是在转录水平上介导的，并定位于SAR1A启动子中的元件。本研究的目的是评估SAR1A基因启动子的多态是否与Hb F水平或HU治疗反应相关。我们研究了386例镰状细胞病患者，其中包括269名接受或不接受HU治疗的成年人和117名新生儿，这些新生儿患有通过新生儿筛查程序确定的镰状细胞疾病。在SAR1A中发现了20个点突变，包括一个非同义突变，其中包括9个以前在SNP数据库中报道的突变。Rs2310991在5UTR型和+31T&gt；C型在5UTR型和+31T&gt；C型中的分布频率差异有统计学意义(优势比OR=1.90，95%可信区间CI=1.1~3.2，P=0.009)和+31T&gt；C(奇比OR 9.81.3~73.9；可信区间CI 95%；P&lt；0.001)。在HU治疗的HB SS患者中，上游5非编码区的3个未知SNP(-809C&gt；T、-502G&gt；T和-385C&gt；A)与HBF应答显著相关(P&lt；0.05)。我们的数据提示SAR1A基因多态性可能参与调节HBF的表达，并调节镰状细胞病患者对HU的反应。

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.