人UREB1基因克隆及其在肿瘤组织中的分布

Molecular Cloning of Human UREB1 and Its Distribution in Tumor Tissue

目的：大鼠 ＵＲＥＢ１基因编码的蛋白质能特异性地结合位于强吗啡基因启动子上游的一段 ＤＮＡ序列（ｕｐｓｔｒｅａｍ ｒｅｇｕｌａｔｏｒｙ ｅｌｅｍｅｎｔ，ＵＲＥ）。早期研究证实 ＵＲＥＢ１对强吗啡基因启动子的转录具有促进作用，而对ｐ５３的转录激活作用具有抑制效应。本实验目的就是克隆人ＵＲＥＢ１基因，探索其与肿瘤发生发展的关系。方法：利用人工合成寡核苷酸探针从人脑ｃＤＮＡ文库中筛选人ＵＲＥＢ１基因，应用大肠杆菌表达的重组蛋白质制备的抗体检测肿瘤组织中ＵＲＥＢ１的表达分布。结果：获得了人ＵＲＥＢ１基因，核苷酸序列在对应区域及氨基酸序列与大鼠ＵＲＥＢ１基因ｃＤＮＡ序列和氨基酸序列皆有９１％的同源性。在各种肿瘤组织中都有ＵＲＥＢ１的表达，但是表达水平及定位不一样。初步发现有这样一个规律，随着肿瘤的恶性程度增加 ＵＲＥＢ在１核内的聚集程度增加。 ＵＲＥＢ１的酪氨酸磷酸化分析结果显示，肿瘤恶性程度高，ＵＲＥＢ１的酪氨酸磷酸化程度高。结论：ＵＲＥＢ１可能参与肿瘤的发生发展，其酪氨酸磷酸化水平可以影响肿瘤的恶性程度。

Objective: Rat UREB1 protein coded by the gene UREB1 can specially bind to URE (upstream regulatory element) which is in the upstream of the promoter. It's reported that the protein of UREB1 promote the transcription of Dynorphin gene and inhibits p53 transactivation. This study was designed to clone human UREB1 gene and explore the relationship between UREB1 and the development of tumor. Methods: The artificial synthetic oligonucleotide was used as the probe to screen human brain cDNA library and human UREB1 gene was cloned. The antibody, which was produced using the recombinant UREB1 from E. coli as the antigen and immunizing the animals, was utilized for detecting the distribution of UREB1 in different tumor tissues. Results: The human UREB1 gene was cloned by using in situ hybridization for screening human brain cDNA library, and the nucleotide sequences and the deduced amino acid sequence of human UREB1 has 91% homology with that of rat UREB1 identified previously. Western blot analysis revealed that the human UREB1 was present in all tumor tissues but the quantity of UREB1 in different tissues was not the same. Immunohistochemistry results shown that the human UREB1 distributes primarily in the cytoplasm and nuclear of tumor cells and nuclear UREB1 in carcinosarcoma is much more than that in adenoma. After analyzing the level of tyrosine phosphorylated UHEB1 in a few tumor tissues, the result shown that the more malignant the tumor tissue was, the higher level the tyrosine phosphorylated of UREB1 was in that tumor tissues. Conclusion: Human UREB1 may be involved in the development of tumor and its tyrosine phosphorylation may affect the degree of tumor malignant.