【作者】 张军哲；

【导师】 覃扬；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 背景与目的DNA甲基化是指在DNA甲基转移酶(DNAmethylatransferase，DNMT)作用下，以S-腺苷蛋氨酸(s-adenosyl-L-methionin，SAM)为甲基供体，将甲基基团转移到胞嘧啶残基C5上。哺乳动物的甲基化修饰通常发生在CpG岛，CpG岛指观测值／期望值大于0.6并且GC含量大于50％的200bp以上的序列区域。哺乳动物基因组中60％～90％的CpG都被甲基化，未甲基化的CpG成簇地组成CpG岛，位于结构基因启动子的核心序列和转录起始点。有研究发现，肿瘤组织中的DNMT，尤其是DNMT1表达水平增高，提示DNMT与肿瘤的异常甲基化相关。谷胱甘肽转移酶P1(glutathloneS—transferase P1，GSTP1)基因定位于染色体的11q13，该基因全长约3kb，包括6个内含子和7个外显子，cDNA序列全长0.633kb，编码210个氨基酸，是GSTs超基因家族中π家族的成员。GSTπ类酶属于体内Ⅱ相代谢解毒酶同工酶家族，在保护机体细胞不受毒物和致癌物基团的损害方面起着重要作用。在多种人类肿瘤中存在GSTP1启动子区CpG岛的甲基化改变，如在乳腺癌和非小细胞肺癌中，普遍存在启动子区的高甲基化状态；GSTP1“CpG岛”甲基化发生在90％以上的前列腺癌DNA标本和70％的前列腺上皮肉瘤DNA标本；在人原发性肝癌(HCC)的研究中，Tchou等发现90％的HCC组织中GSTP1多肽表达缺失，85％的HCC中GSTP1启动子区高度甲基化。Zhong等发现HCC组织中GSTP1的CpG岛高度甲基化，同时检测到肝细胞株HepG2和Hep3B中GSTP1均为甲基化状态且表达缺失，用甲基化酶抑制剂处理后表达均恢复。作为抑癌基因，GSTP1是一重要的DNA损伤修复基因，其基因启动子区甲基化与多种肿瘤易感性相关。因此，通过药物干预，逆转甲基化的GSTP1启动子区有望成为肿瘤治疗的新策略。在本室前期初步验证中药成分CDP具有抑制多种组织来源的肿瘤细胞的生长，并呈浓度、时间依赖性的基础上，我们将进一步研究其逆转高甲基化状态的GSTP1基因启动子低转录活性的作用并探讨其作用机理。方法含非甲基化的GSTP1启动子区的重组质粒pGL3-GSTP1pro由本实验室前期构建。利用KpnⅠ、XhoⅠ将pGL3-GSTP1pro质粒双酶切，胶回收GSTP1启动子区片段，在SssⅠ甲基化酶的作用下将所有CpG位点甲基化，并用HpaⅡ酶切鉴定甲基化的完整性。完全甲基化的GSTP1启动子区片段与载体利用T4连接酶进行连接，获得含完全甲基化的GSTP1启动子区的重组质粒pGL3-GSTP1pro(?)。利用转染试剂将pGL3-GSTP1pro(?)转入人乳腺癌细胞系(MCF-7)中，同时以去甲基化药物CDP和5-aza-C作用细胞，检测表达的萤光素酶活性。观察GSTP1启动子转录活性的变化。结果重组质粒pGL3-GSTP1pro(?)经KpnⅠ、XhoⅠ双酶切得到的完全甲基化的GSTP1启动子区片段，用对甲基化敏感的HpaⅡ酶切鉴定，经修饰的片段长度酶切后无改变，表明重组质粒构建成功。药物处理后72h，启动子区转录活性明显升高，10μmol／L、30μmol／L、50μmol／L CDP给药组，随药物浓度增大，转录活性依次升高，呈剂量依赖性的关系。(非给药组，相对荧光素酶活性均值为6.35，10μmol／L、30μmol／L、50μmol／L CDP给药组相对荧光素酶活性均值分别为18.23、21.75、24.83)。结论通过构建甲基化GSTP1启动子荧光素酶报告基因载体并转染MCF-7细胞，证实中药成分CDP能够逆转高甲基化状态的GSTP1基因启动子区的低转录活性。更多还原

【Abstract】 Background and object DNA methylation is catalyzed by specific DNA methyltransferases (DNMTs), with S-adenosyl-L-methionine (SAM) as the methyl donor. In mammals, the major target for DNA methylation is the cytosine located to a guanine (5’-CpG-3’). These targets of methylation are not equally distributed in the genome, but found in CpG islands. CpG islands are sequences longer than 200bp with a GC content of over 50% (in contrast to a genome-wide average of about 40%) and an observed over expected ratio of 0.6 or greater of CpG. In mammals cells, approximately, sixty percent to ninety percent of CpG island are methylated. Unmethylated CpG islands are found mainly in the 5’-regions of housekeeping genes as well as some other tissues specifically expressed genes and usually extend from the promoter region into the first exon and sometimes into intron 1. Increased expression of DNMTs has been reported in human cancer, Nonetheless, DNMT1 has been more prominently implicated in cancer development than other DNMTs. Glutathione S-transferases p1 (GSTP1) is an autosomal gene located at chromosome 11q13 within 30kb, including 6 introns and 7 exons, and encoding 210 amino acid. GSTP1 belongs to a family of GSTs isoenzymes, that defend cells against damage mediated by oxidant and electrophilic carcinogens. It catalyzes the conjugation of glutathione with electrophilic compounds, including carcinogens and exogenous drugs, resulting in less toxic and more readily excreted metabolites. AS a Tumor suppressor gene, the CpG island encompassing the GSTP1 becomes hypermethylated associated with pathogenesis of many human cancers. During the pathogenesis of human hepatocellular carcinoma (HCC), 90% HCC tissues fail to express GSTP1 mRNA or GSTP1 polypeptides, 85% HCC tissues in which the CpG island encompassing the GSTP1 becomes hypermethylated. HepG2、Hep3B cells, the human HCC lines, have been shown to contain densely hypermethylated GSTP1 CpG island sequences and to be devoid of GSTP1 mRNh. Treatment with DNMT inhibitor has been reported to trigger the reactivation of genes carrying somatic CpG island hypermethylation.Therefore, restoration of "silenced" gene expression via therapeutic reversal of CpG island hypermethylation may be considered for use as cancer chemoprevention drugs as well as cancer treatment drugs.In previous study of our laboratory work, inhibition of growth of cancer cells derived from different tissues by CDP has been demonstrated in a dose, time-dependent mode. On this basis, further study on reactivation of hypermtehylated GSTP1 Promoter activity in MCF-7 Cells by CDP and explanation of mechanism was carried on in present work. Methods Recombinant plasmid of pGL3-GSTPlpro~m that contains hypermethylated GSTP1 promoter were constructed and then transiently transfected into human breast cancer cell line MCF-7 cell. After treated with CDP and 5-aza-C, The luciferase activity in lysates were assayed.Results The GSTP1 promoter DNA fragment was modified with the CpG methylase, M. SssⅠ, identified with the HpaⅡ, an sensitive restriction enzyme to methylation. The methylated GSTP1 promoter DNA fragment was not cleaved. It suggested that The recombinant plasmid pGL3-GSTPlpro~m with methylated GSTP1 promoter was successfully constructed. Low promoter activities were found in hypermethylated GSTP1 promoter. The promoter activities were reactivated and in a dose-dependent mode.Conclusion CDP has the ability to reactivate the hypermethylated GSTP1 gene promoter activity.更多还原