人肝癌低表达基因MT1M的克隆和功能研究

【作者】 孙路虹；

【导师】 张锡然； 余龙；

【作者基本信息】 南京师范大学 ， 动物学， 2003， 博士

【摘要】 金属硫蛋白(Metallothionein,MT)是一类富含半胱氨酸的低分子量蛋白。除了参与金属代谢、解毒以及清除羟基自由基之外，该家族成员与各种肿瘤的关系越来越引起人们的重视。肝脏是人体多能性器官，具有解毒，物质合成，调节营养代谢平衡的功能。一些疾病如：肝癌，乙肝，肝硬化会引起肝功能的失调。肝细胞癌(Hepatocellular Carcinomas,HCC)是我国人群中高发的一类恶性肿瘤，尤其是我国的东南部。寻找肝癌相关基因并研究其生物学功能对于了解肝癌的发生发展、揭示肝癌的发生机制、早期诊断和治疗有着重要的理论和实际意义。 在对肝细胞与肝癌细胞的差异表达研究中，我们得到了一条300bp的cDNA，它在肝癌细胞中的表达显著下调，以此cDNA序列为探针，我们在NCBI数据库中筛查EST文库，拼接出447bp的全长cDNA序列。其开放阅读框编码61个氨基酸，在阅读框两侧设计引物，经PCR扩增，测序，证实序列一致。此外，基因组序列也经PCR扩增，测序验证，序列长1753bp，包括三个外显子和两个内含子，与人金属硫蛋白家族其他同源基因结构类似。蛋白质编码分析是金属硫蛋白，经查新验证发现为一个新的基因，即金属硫蛋白家族的新成员，将该基因送国际基因命名委员会并被命名为MT1M。生物信息学分析MT1M位于人的第16号染色体。利用人16种组织的杂交膜对MT1M基因的组织表达谱进行了检测，Northern杂交结果显示在肝、肾和胰腺组织中高表达，在心脏、骨骼肌、前列腺、小肠和结肠组织中呈中度表达，而在脾、胸腺、睾丸、卵巢、外周血淋巴细胞、脑、胎盘和肺组织中呈低表达。为了探讨MT1M与肝癌发生的关系，我们首先用Northern Blot方法检测8对肝癌及癌旁组织及2例胎肝组织，结果显示8对肝癌及癌旁组织中有7对呈现下调表达，而2例胎肝组织则无明显变化。58对肝癌及癌旁组织的半定量RT-PCR分析显示有51对呈现下调表达。结果提示MT1M基因与肝癌的发生有密切关系。 通过构建原核表达载体，我们在原核表达系统中对MT1M基因进行了重组表达并获得了纯化的蛋白。以重组蛋白为抗原我们制备了兔源的多克隆抗体，并用它进行肝癌及癌旁组织的免疫组化研究。从蛋白水平证明MT1M的表达是在癌旁组织，肝癌组织则呈下调表达，并且定位在细胞浆。我们通过Northern杂交和RT-PCR检测了肝癌细胞株中MT1M基因的表达，在L-02，Hep-G2，YY-8103，SK-hep-1，Focus，Huh-7，Hep-3B，BEL-7402及QGY-7703九株肝癌细胞株中，半定量RT-PCR检测MT1M基因的表达结博士学位论文 人肝癌低表达基因MT M的克隆和功能研究果：Hub司表达量最高；HepGZ次之；随后是 YYSI 03，SK人ep、Focus、L02和QGY7703；BEL－7402和Hep3B表达量很低。Northern杂交检测结果与半定量RTPCR检测结果基本一致。用 Northe。杂交检测显示低剂量 As。O。可诱导部分肝癌细胞株MT基因的表达。同时我们用MTS方法观察不同剂量的三氧化二砷（AS203）在不同时间（24h、48h、72h）对这些肝癌细胞株的毒性作用。结果表明，ASZO；在一定浓度范围内，L02，Hep－GZ细胞几乎不受AszO3的影响，对Hep｝B、BEL刁402、Hub7细胞有作用；对 SK人eP、YYSI 03细胞作用较强，即不同的肝癌细胞株对药物的敏感性有所不同。为了进一步探讨 MT与肝癌之间的关系，我们构建了真核表达载体并转染肝癌细胞株，观察TMTIM基因在此p－GZ细胞中可能参与的信号通路。用MTIM基因瞬时转染HepGZ细胞检测NFkB和AP的转录，结果发现MTIM能促进NFkB转录活化；影响 NF一肥信号通路；对 API活化的 WK信号通路无明显影响。提示 MTIM介导NF一肥的抗凋亡作用。 本研究首次克隆了一个金属硫蛋白家族的新成员 MT，并在基因水平、蛋白水平和细胞水平探讨了其与肝癌之间的关系，这些结果提示肝癌的发生机制是一个涉及到众多领域的复杂过程，我们的研究仅从某一个方面揭示MTIM与肝癌发生的相关性，同时MTIM表达量不同的肝癌细胞株对ASZO3的敏感性有所不同，提示MTIM可能作为指导II＄床用药的标志之一。有待进一步研究验证。更多还原

【Abstract】 Metallothioneins (MTs) are short, cysteine-rich proteins for metal metabolism and detoxification; they act as radical scavengers and are related to various human tumors. Liver is an important multifunctional organ performing detoxification, substance synthesis and metabolic balance of nutrients. However some diseases such as hepatoma, hepatitis B, cirrhosis may distort it’s function. The satisfactory treatment of these diseases is yet to come and will depend largely on the further study of genes involved in the functions of liver. Liver cancer is one of the most common malignant tumors in China, especially in Southeast China. Seeking liver cancer-related genes and characterizing their biological functions will help to reveal the mechanism of liver tumorigenesis , and diagnose and treatment early.In our present study, grounded on a differentially displayed cDNA fragment down-regulated in hepatoma tissues, a series of human ESTs homologous with the cDNA fragment were obtained and assembled into EST contigs. These EST contigs were sent to compare with the database of NCBR division of GenBank, and only one contig sequence with 474bp was proved to be novel gene sequence. The reliability of the contig sequence was subsequently confirmed by PCR amplification on human liver cDNA library with the gene-specific primers and sequencing. The cDNA contains an ORF coding for a peptide of 61 amino acids showing great similarity to the known MT1 family sequences. This cDNA was therefore deposited in GenBank database and later named MT1M by the Human Nomenclature Committee.In addition, the genomic DNA sequence of MT1M was determined by performing PCR on human genomic DNA library with the same gene-specific primers and sequencing. The results showed that the gene possessed of two introns and three exons, and shared identical exon/intron boundaries with all the other mammalian MTs. MT1M gene was localized in the 16 chromosome q13. MT1M was widely expressed in all 16 adult tissues tested with Northern Blot analysis. The 0.5Kb transcript was most abundant in liver, kidney and pancreas, moderate in heart, skeletal muscle, prostate, small intestine, and colon, and very weak in spleen, thymus. testis, ovary, peripheral blood leukocyte, brain, placenta, and lung. It was interesting to find that MT1M was strongly expressed in normal adult and fetal liver tissues, but weakly inhepatocellular carcinoma tissues (7 of 8 cases) by northern blot analysis. The decreased expression of MT1M in HCC was further supported by multiplex semiquantative RT-PCR, which showed that MT1M was significantly down-regulated or absent in 51 of 58 cases.Recombinant gene of MT1M was constructed to pGEX-4T vector and was expressed in E coli (BL21) system. Purified recombinant protein is 32KD as expected and was used as antigen to make polyclonal antibody in New Zealand rabbits. The polyclonal antibody was applied to immunohistochemical research. MT1M also expressed strongly positive in the matched adjacent tumor-free tissues, whereas negative staining in HCC tissue The results of Northern blot analysis and multiplex semiquantative RT-PCR revealed that MT1M was variably expressed in human hepatoma cell lines. It was found that the expression level of MT1M was high in Huh-7, HepG2, moderate in YY-8103, SK-PEP-1, L-02, Focus , QGY-7703, low in BEL-7402, Hep3B. The expression of MT1M was indused in some hepatoma cell lines by arsenic trioxide at low doses. The hepatoma cell lines were exposed to different doses of the reagents for 24h, 48h and 72h, the cytotoxicity of arsenic trioxide was determined by MTS assays. The viability of HepG2 and L-02 cells was hardly affected by the treatment of arsenic trioxide, whereas the viability of SK-PEP-1, BEL-7402, YY-8103, Huh-7and Hep3B cells decreased as the concentration of the treatment of arsenic trioxide was increased. Besides, MT1M activated the NF-kB-depentent transcription, as measured by dual luciferase assay in human HCC cells HepG2 transfected with NF-kB -responsive reporter construct and API -responsi更多还原