【作者】 董辉；

【导师】 赵国屏；

【作者基本信息】 复旦大学 ， 微生物学， 2010， 博士

【摘要】 原发性肝细胞癌(Hepatocellular carcinoma, HCC)是全球最常见的恶性肿瘤之一,在中国更是位居肿瘤致死性疾病的第二位,严重危害了我国人民健康。多年来科学家们在HCC的基础和临床研究中取得了显著成就,但由于HCC的高度异质性,还需要更多的研究来进一步加深对其致病分子机理的认识和理解。本研究中,我们从寻找HCC差异表达基因和基因组DNA水平异常改变两方面入手,对HCC发生发展的分子机制进行了探讨。在第一部分研究工作中,我们应用SAGE和LongSAGE技术构建了正常肝组织、HepG2细胞和HCC的基因表达谱,其中用于构建HCC的SAGE文库和LongSAGE文库的组织标本来自于同一个病人。通过对HepG2细胞与正常肝组织的基因表达谱的比较分析,我们发现在HepG2细胞中差异表达的基因主要涉及编码核糖体蛋白的基因、MAPK信号传导通路基因、细胞周期相关基因、细胞粘附相关基因、钙离子信号传导通路基因、凝血和补体功能相关的基因以及烟酸／烟酰胺代谢相关的基因。比较HCC与正常肝组织的基因表达谱的差异,我们发现在HCC中差异表达的基因主要包括细胞合成、细胞增殖与死亡、信号传导、物质转运、凝血功能、细胞代谢以及细胞对外界刺激发生反应等生物过程相关的基因。选取部分差异表达基因进行验证,结果表明,PEG10、SGCE、ZNF83、PTPN12和TM4SF1基因等12个基因在肝癌组织中的表达高于相应的癌旁组织,差异具有显著性(p<0.05)。值得注意的是,SGCE和PEG10基因共同位于7q21区域,两基因的5’端仅相距115bp,并且两基因在75%的HCC病例中同时发生表达上调或下调,以上调为主。在第二部分研究工作中,我们借助Digital karyotyping技术和Genome Sequencer FLX超高通量第二代测序系统,绘制了HCC全基因组DNA异常改变的高分辨率图谱。我们将Genome Sequencer FLX测序系统自备的平端接头改造为粘端接头,从而保留了基因组序列标签串联体的粘性末端所携带的信息,提高了测序效率。Digital karyotyping文库总共测序111,585条,得到358,931个有效序列标签。通过对序列标签数据的分析,发现在HCC中发生扩增的染色体主要有1、4、6、7、8、9、12、15、17、19、21、X和Y染色体,发生扩增的染色体区域内包含了BAGE、UTRN、DYNC1I1、SLC25A13、POTEB和CEACAM21等多个基因。其中DYNC1I1和SLC25A13基因在染色体上的位置邻近PEG1O和SGCE基因,也位于7q21区域。进一步的研究发现,DYNC1I1、SLC25A13、POTEB、CEACAM21、PEG10和SGCE基因分别在36%、52%、50%、36%、23%和32%的HCC病例中出现基因组DNA的扩增,且PEG10、SGCE、DYNC1I1和SLC25A13基因在HCC中的表达显著高于相应的癌旁组织(p<0.05)。在55-69%的HCC病例中,PEG10、SGCE、DYNC1I1和SLC25A13基因组DNA的扩增同时伴有基因表达水平的上调。这些结果提示,位于7q21区域的4个基因—PEG10、SGCE、DYNC1I1和SLC25A13可能共同参与了HCC发生发展的分子机制,而基因组DNA的扩增可能是这些基因表达上调的原因之一酪氨酸磷酸酶的异常失活或激活,导致蛋白质分子中酪氨酸残基的磷酸化水平异常升高或降低,是HCC的重要生物学特征之一。在第三部分研究工作中,我们应用酵母双杂交技术,对受体型酪氨酸磷酸酶PCP-2相互作用蛋白进行了初步研究。通过筛选人脑组织酵母cDNA文库,找到可能与PCP-2发生相互作用的蛋白质—衔接蛋白3(adaptor protein-3, AP-3)的p3A亚基,并在哺乳动物细胞中进一步验证了两者之间的相互作用。AP-3的β3A亚基可能通过识别PCP-2胞浆区的YXXΦ模体和／或LL模体与PCP-2结合,从而参与PCP-2细胞内转运、定位、以及受体的循环再利用等过程。我们通过对HCC基因转录水平和基因组DNA水平异常改变的研究,进一步加深了对HCC致病分子机制的认识,为今后选择可用于HCC诊断、病程预测、转归和预后判断的分子标记物,以及分子靶向药物治疗的作用靶点提供了新的理论依据。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and the second cancer killer in China, threatening Chinese people’s health severely. Significant achievements have been made in basic and clinical research of HCC. However, as HCC is a kind of highly hetero-geneous disease, more studies are needed to deepen the cognition and un-derstanding of molecular mechanism of hepatocarcinogenesis. In this study, we try to elucidate the molecular mechanism more clearly by searching for genes differentially expressed in HCC as well as the abnormal alterations in HCC genomic DNA.In the first part, we applied SAGE and LongSAGE to obtain the gene ex-pression files of normal liver, HepG2 and HCC. The same HCC sample was further analyzed simultaneously using LongSAGE. By comparing gene ex-pression profiles of HepG2 and normal liver, we found that genes differentially expressed in HepG2 included those encoding ribosomal protein, blood coa-gulation factors, complement factors, as well as those involved in MAPK sig-naling pathway, cell cycles regulation, cell adhesion, calcium signaling pathway, and nicotinic acid/nicotinamide metabolism. Comparison of gene expression profiles of HCC and normal liver leaded to the identification of genes differen-tially expressed in HCC, which participated in biological processes of biosyn-thesis, cell proliferation, cell death, signal transduction, transport, blood coa-gulation, response to external stimulus, and cellular metabolism. Totally 12 genes (including PEG10, SGCE, ZNF83, PTPN12, TM4SF1 et al) differently expressed in HCC identified by SAGE were confirmed to be up-regulated in HCC compared to the corresponding nontumorous liver (p< 0.05). Among these 12 genes, it was worth mentioning that SGCE and PEG10 locate to-gether within 7q21, separated by only 115bp base pairs between the 5’ ends of these two genes. Furthermore, similar expression patterns (up-or down-regulated simultaneously) of these two genes were observed in 75% of HCC cases detected, being up-regulated together in most cases.In the second part, we obtain a comprehensive and high resolution map of abnormal alterations in HCC genomic DNA with the help of Digital karyotyping technology and ultrahigh-throughput second generation sequencing system of Genome Sequencer FLX. The blunt-ended adaptors provided by Genome Sequencer FLX was modified to keep critical information contained in the ad-hesive ends of genomic tag concatemers, which in turn improved sequencing efficiency. Totally 111,585 reads were sequenced and 358,931 filtered tags were obtained from the Digital karyotyping library. By analyzing distribution of these filtered tags on each chromosomes, we found that genomic DNA ampli-fication in HCC were mainly occurred on chromosome 1,4,6,7,8,9,12,15, 17,19,21, X and Y. Genes involved in the amplified regions included BAGE, UTRN, DYNC1I1, SLC25A13, POTEB and CEACAM21 et al. The DYNC1I1 and SLC25A13 genes also locate on 7q21, very close to PEG10 and SGCE. Genomic DNA of DYNC1I1, SLC25A13, POTEB, CEACAM21, PEG10 and SGCE were detected to be amplified in 36%,52%,50%,36%,23%and 32% of HCC cases respectively. Gene expression level of DYNC1I1, SLC25A13, PEG10 and SGCE were also found to be increased in HCC compared to the corresponding nontumorous liver (p< 0.05). For DYNC1I1, SLC25A13, PEG10 and SGCE genes, amplification of genomic DNA accompanied by up-regulation of corresponding genes were observed in 55-69% of HCC cases. These results indicated that DYNC1I1, SLC25A13, PEG10 and SGCE genes located together within 7q21 may be involved in molecular mechanisms of development and progression of HCC, and genomic DNA amplification may play a role in their up-regulation in HCC.One of the most important biological characters of HCC is the improper activation and inactivation of protein tyrosine phosphatases which leads to abnormal alteration in the phosphorylation level of tyrosine amino acid. In the third part, we performed preliminary study on finding potential proteins inte-racted with PCP-2, a member of protein tyrosine phosphatases, using yeast two-hybrid technology. By screening the pre-transformed yeast library of human brain cDNA, the interaction between PCP-2 and theβ3A subunit of adaptor protein (AP)-3 was detected in yeast and further confirmed in mam-malian cells. Theβ3A subunit of AP-3 may interact with PCP-2 through rec-ognition of YXXΦand/or LL motifs contained within cytoplasmic domain of PCP-2, thus participating in biological processes of intracellular trafficking, lo-calization or recruitment of PCP-2.Our study on the transcriptome and genomic DNA abnormalities in HCC may lead to better understanding of molecular mechanism of hepatocarcino-genesis, providing more potential molecular markers for diagnosis, progres-sion, metastasis and prognosis of HCC, as well as more potential targeted genes for designing molecular drugs for HCC therapy.更多还原