【作者】 杨诏旭；

【导师】 窦科峰； 赵忠良；

【作者基本信息】 第四军医大学 ， 外科学， 2005， 博士

【摘要】 Gankyrin是近年来在肝细胞癌中发现的一个新的致癌基因编码的蛋白,因其几乎在所有的肝癌患者的癌组织中均存在过表达而受到重视。Gankyrin蛋白可以与p16^INK4A竞争结合Cdk4,从而对抗p16^INK4A对Cdk4的抑制作用,进而对抗了p16^INK4A对肿瘤细胞增殖的抑制作用。Gankyrin的表达升高可以引起Rb磷酸化水平上升,导致转录因子E2F1的活性释放,引起细胞增殖的一系列相关基因的转录激活。此外由于gankyrin蛋白存在于26s蛋白酶体的19s调节复合体中,且可与Mdm2发生相互作用,而Mdm2属于泛肽连接酶,介导泛素/26s蛋白酶体对P53的降解,因此认为gankyrin可能参与了P53的降解过程。p16^INK4A、Rb、P53等的失活目前被认为是肝癌发生过程中最为关键的几个因素,而gankyrin则同时参与在三个过程之中。近来有实验在Fischer 344鼠体内诱发肝癌,结果发现在肿瘤发生的最初阶段,即出现了gankyrin的表达升高,这些均提示我们gankyrin可能是肝癌发生发展过程中的一个关键基因,同时也为肝癌的治疗提供了一个潜在的靶更多还原

【Abstract】 Gankyrin is a novel gene identified from human hepatocellular carcinoma,which is overexpressed in almost all studied hepatocellular carcinomas. Gankyrin binds to Cdk4 and counteracts the inhibitory function of the tumor suppressors p16~INK4A. Overexpression of gankyrin increases both the phosphorylation and degradation of Rb.And inactivation of RBI by phosphorylation seems to release E2F from an inhibitory complex, enabling it to promote the transcription of genes necessary for progression into late G1 and S phases.Because gankyrin protein is one of the non-ATPase subunits of 19 S complex,which is a 700 kDa multisubunit regulatory complex of the human 26 S proteasome,and gankyrin can interact with Mdm2 ,which is an E3 ubiquitin ligase involved in p53 degradation, So gankyrin is thought to be related with the degradation of p53.It was reported that the inactivation of some tumor suppressive genes such as p53 ,pl6~INK4Aand Rb is critical to tumorigenesis in HCCs,and gankyrin seems to be involved in all three progression.Recently,researchers induced hepatocellular carcinoma in Fisher 344 rats and observed which genes involved in the tumorigenetic progress,the results showed that gankyrin is overexpressed from the earliest stage of tumor development. These findings suggest that gankyrin is a major player in cellcycle control and tumorigenesis in HCCs.lt also provided us a promising target for therapy.The present study consists of three parts:Part One: The expression of gankyrin gene in hepatocellular carcinoma cells and the gene’s clone, prokaryotic expression and antibody preparationAIM: To determine the expression of gankyrin gene in hepatocellular carcinoma cell lines . To express gankyrin fusion protein in E.coli and to prepare antibody against it. METHODS:The expression of gankyrin in HepG2,SMMC-7721 and HHCC cell lines was detected using RT-PCR analysis. The core fragment of gankyrin was cloned into plasmid pGEX-4T-2 containing glutathione s-transferase(GST) fusion protein gene and plasmid pRSET-B containing His fusion protein.Following restriction enzyme digestion analysis and sequencing.Reformed plasmid was transformed into E.coli DH-5a. Gankyrin fusion protein was expressed under IPTG induction .Then the purified gankyrin protein was used to immunize New Zealand rabbits.The specific antibody against Gankyrin was identified by ELISA and Western blot. RESULTS: The expression of gankyrin mRNA and protein could be detected in all three hepatocellular carcinoma cell lines. pGEX-4T-Gankyrin and pRSET-B-Gankyrin was constructed successfully,but only GST-Gankyrin fusion protein was expressed in E.coli and it specific polyclonal antibody was obtained. CONCLUTION: Gankyrin gene expressed in HepG2,SMMC-7721,HHCC cell lines. The successful expression of GST-Gankyrin fusion protein in E.coli and the preparation of it’s specific polyclonal antibody will be valuable for the study of the biological function of gankyrin.Part Two: Suppressing the expression of gankyrin in human hepatocellular carcinoma cell by RNAiAIM: To construct the plasmid containing short hairpin RNA (shRNA)targeting at gankyrin in order to inhibit the expression of gankyrin in HepG2 cell lines. METHODS: Three couple of shRNA sequence corresponding to gankyrin were synthesized and inserted into plasmid pGenesil-1 under U6 promoter to construct recombinant plasmid pGenesil-1 -Gankyrin 1 ^ pGenesil-1-GankyriiL^ pGenesil-l-Gankyrin3, and the vector expressing shRNA which is irrelevant with all human gene was used as control (pGenesil-1- control) ,then HepG2 cell lines stably transfected with them were established and gene expression inhibition was assessed by RT-PCR and Western blotting analysis. RESULTS: The recombinant plasmid pGenesil-1-Gankyrin 1 > pGenesil-1-Gankyrh^ pGenesil-l-Gankyrin3 and pGenesil-1- control were successfully constructed. PGenesil-1-Gankyrin2> pGenesil-l-Gankyrin3 suppress the expression of gankyrin significantly in HepG2. CONCLUTION: The result showed the shRNA of gankyrin can efficiently inhibited the expression of gankyrin in HepG2. RNAi mediated by shRNA is a useful methods in gankyirn gene silence.Part Three: Effects on proliferation of suppressing the expression of gankyrin in human hepatocellular carcinoma cell line HepG2AIM: To explore the effect on morphology,proliferation , cell cycle and tumor formation in nude mice when suppressing gankyrin expression in HepG2 cell line. METHODS: Observe the cells stably transfected with different plasmid under electron microscope.The inhibitory effect on cell growth was studied by cell counting and MTT assay, and cell cycle was analyzed by flow cytometry. Then tumor formation experiment was carried on nude mice.RESULTS: Compared with the cells transfected with control, electron microscopy show the cell has lower ratio between nuclear and plasma,smaller nucleolus and lower activity when suppressing the expression of gankyrin.And the growth of HepG2 cell was inhibited significantly , the number of the cells was much lower,MTT assay showed cell proliferation index decreased,flow cytometric analysis demonstrated that the cell cycle was stucked at Gl phase. Suppressing gankyrin also inhibited tumor formation in更多还原