三个肝癌相关基因的初步研究

【作者】 陈剑；

【导师】 余龙；

【作者基本信息】 复旦大学 ， 遗传学， 2005， 博士

【摘要】 肿瘤发生机制的研究一直是肿瘤分子生物学中的一个重要研究领域,其中肿瘤相关基因的克隆和鉴定,不仅对肿瘤机制的探讨具有重要的理论价值,而且对肿瘤的诊断和治疗具有潜在的应用价值。肝癌是亚洲和非洲一些国家常见恶性肿瘤之一,肝癌的发生机制现在还不清楚,被认为是多步骤多因素作用的结果,认为与癌基因的激活和抑癌基因的失活等基因背景有关系。鉴定癌旁组织与肝癌表达有差异的基因可为肝癌的预防、诊断和治疗研究提供有价值的候选基因。 一、Cyclophilin（CYPs）属于脯氨酸异构酶家族,能够催化脯氨酰多肽和脯氨酰蛋白的顺反结构互变,并能够和环孢菌素A结合。现在发现CYP亚家族不但在免疫抑制方面发挥重要作用,而且在信号传递、细胞凋亡、RNA剪接、细胞有丝分裂以及肿瘤发生、HIV病毒感染等方面都有重要作用。本文第一章研究结果是根据CYP家族的保守性克隆了人、小鼠和大鼠的CYPJ基因。检测原核表达的CYPJ蛋白发现有PPI酶的活性,测得k_cat=26.9s^-1,K_m=649.6μM,k_cat/K_m=4.14×10⁴M^-1S^-1。CsA可以抑制酶活性。并且发现CYPJ基因的mRNA水平在71.42%的肝癌组织中上调;统计分析发现CYPJ上调表达与肝癌Edmondson’s分级有显著相关;大鼠的CYPJ基因在肝脏中胎鼠时表达高,成熟后降低;在睾丸中则随性成熟睾丸发育而表达增高。其次发现CYPJ基因可以促进LO2细胞克隆形成,CYPJ稳定表达株细胞接种于裸鼠皮下其生长速度较对照生长加快。CYPJ基因瞬时转染可以引起细胞G1期减少,S期增加。酵母双杂交筛选到SLU7,ARL6IP4等基因产物可以和CYPJ相互作用。SLU7,ARL6IP4都与pre-mRNA剪切有关。 二、脯氨酸异构酶家族的另一个成员Pin1也被认为与肿瘤有关。Pin1调节与细胞周期有关的几个重要蛋白质,其中NIMA（Aspergillus nidulans）是第一个被鉴定的与Pin1作用的蛋白。Nek6是NIMA在人类的同源基因。在本文第二章中我们用GST pull-down、免疫共沉淀和免疫荧光共定位实验证明Pin1与Nek6能够相互作用。并且Pin1在62.%的肝癌组织中高表达,而Nek6则在70%的肝癌组织中高表达。统计分析显示两者在肝癌组织中的表达有正相关性。 三、λ-晶体蛋白作为眼球晶体组织中的结构成分只在家兔和野兔的晶体中存在。在晶体之外的组织中存在则发挥其他方面的作用。人CRYL1基因是兔λ-更多还原

【Abstract】 Study on carcinogenesis has always been an important research area in tumor molecular biology. Cloning and identification of tumor-related genes have both theoretical and clinical values in inquiring into carcinogenesis, cancer diagnosis and curing. Human hepatocellular carcinoma （HCC） is one of the most common malignant tumors in some countries of Asia and Africa. The pathogenesis of human HCC is thought to involve multiple steps and multiple genes. The pathogenesis of HCC is not yet clear, but it is frequently associated with a background of genetic alterations which includes the activation of oncogenes and the inactivation of tumor suppressor genes. Identification of differential expressed genes between normal and HCC will offer great opportunities of prevention, diagnosis and treatment for HCC.Cyclophilins （CYPs） are a group of proteins having Peptidyl-prolyl cis-trans isomerases （PPIases） activity which catalyses the isomerization between two proline conformations in proteins. They act as chaperons assisting protein-folding events and have been proved to have many other functions in immunosuppression, signal transduction, as well as cell cycle regulation. In this study, based on evolutionary conservation of CYPs, the cDNAs of human, mouse and rat CYPJ genes were cloned. Transformation of the cDNA fragment containing the entire ORF of human CYPJ into E. coli for transient expression generated protein with PPIase activity. Its value of k_cat=26.9s^-1 , K_m=649. 6μM and k_cat/K_m= 4.14 × 10⁴M^-1S^-1. CsA, an immunosuppressive drug that can bind to CYPs, can inhibit the PPIase activity of CYPJ. Interestingly, we found that human CYPJ was highly expressed in fetal liver and mature testis, and was up-regulated in human hepatocellular carcinoma, suggesting an important role of CYPJ in cells with high proliferation and metabolism rates. This confirmed by the test of clone conformation. The critical function of CYPJ was further proved by L02 and SK-Hep-1 cells which stably express CYPJ can accelerat the tumor growth in nude mice. In addition, Cell cycle experiment showed that the cells with overexpression of CYPJ arrested in G1 phase is dramatically reduced, the number of cells in S phase is更多还原