Functional characterization of proximal promoter of gene for human BRAK/CXCL14, a tumor-suppressing chemokine
Functional characterization of proximal promoter of gene for human BRAK/CXCL14, a tumor-suppressing chemokine
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DOI：
10.2220/biomedres.31.123
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发表时间：
2010-04-01
影响因子：
1.2
通讯作者：
Hata, Ryu-Ichiro
中科院分区：
文献类型：
--
作者：
Komori, Reika;Ozawa, Shigeyuki;Hata, Ryu-Ichiro
BRAK/CXCL 14是一种在许多正常细胞和组织中表达的趋化因子，但在转化细胞和癌组织（包括头颈部鳞状细胞癌（HNSCC））中不存在或以非常低的水平表达。我们先前报道了在HNSCC细胞中强制表达BRAK/CXCL 14降低了无胸腺裸鼠和SCID小鼠中肿瘤形成率和肿瘤异种移植物的大小，表明该基因的表达水平对于肿瘤抑制是重要的。为了研究该基因表达的调控机制，我们确定了该基因的转录起始位点和启动子基序。5'端快速扩增cDNA末端法确定该基因的主要转录起始位点位于第一个位点下游283 bp处。荧光素酶活性的报告基因构建体与各种缺失或突变的测定表明，一个非典型的TATA样序列，TATTAA是必不可少的基因的转录和AP-1结合序列和串联GC盒是必要的刺激基因在人类鳞状上皮细胞的表达。人DNA区域与小鼠基因高度同源（95%碱基同一性）。此外，冈田酸，一种丝氨酸/苏氨酸磷酸酶1，2A和2B的抑制剂，刺激TATTAA序列和AP-1结合序列依赖性启动子活性，以及增加BRAK/CXCL 14 mRNA的水平，表明这些序列是必不可少的调节BRAK/CXCL 14基因在细胞中的表达。
BRAK/CXCL14 is a chemokine that is expressed in many normal cells and tissues but is absent from or expressed at very low levels in transformed cells and cancerous tissues including head and neck squamous cell carcinoma (HNSCC). We reported previously that the forced expression of BRAK/CXCL14 in HNSCC cells decreased the rate of tumor formation and size of tumor xenografts in athymic nude mice and SCID mice, suggesting that expression level of the gene is important for tumor suppression. In order to study the regulatory mechanisms governing the expression of this gene, we determined the transcriptional start site and promoter motifs of the gene. The major transcriptional start site determined by 5' rapid amplification of cDNA end method was located 283 bp downstream of the first proposed site of the gene. Determination of luciferase activities of reporter gene constructs with various deletions or mutations showed that an atypical TATA-like sequence, TATTAA was essential for the transcription of the gene and that the AP-1 binding sequence and tandem GC box were necessary for stimulating the expression of the gene in human squamous epithelial cells. The human DNA region was highly homologous (95% base identity) to the mouse gene. In addition, okadaic acid, an inhibitor of serine/threonine phosphatases 1, 2A and 2B, stimulated TATTAA sequence and AP-1 binding-sequence dependent promoter activity as well as increased the level of BRAK/CXCL14 mRNA, indicating that these sequences are essential for the regulation of BRAK/CXCL14 gene expression in the cells.