1.TBP和TLP对hTERT的转录调节作用及机制 2.DNA甲基化在宫颈癌诊断中的应用

【作者】 杨楠；

【导师】 张波；

【作者基本信息】 北京大学 ， 病理学和病理生理学， 2008， 博士

【摘要】 端粒酶的激活是细胞癌变过程中的重要步骤,研究发现多种因素影响细胞中端粒酶的活性。人端粒酶催化亚单位(hTERT)是人端粒酶活性的必需成分,hTERT的表达是端粒酶激活的限速步骤,其水平决定了细胞中端粒酶的活性。研究发现,hTERT基因表达的调节主要发生在转录水平,目前已发现几种转录因子调节hTERT基因的转录,但确切的调控机制有待深入研究。细胞中编码功能蛋白的基因转录已经证明依赖RNA聚合酶II的活性。但是,RNA聚合酶II并不是直接与启动子区结合而是通过细胞蛋白的介导。所以,转录起始的第一步是蛋白识别并物理性的结合启动子区的一些特殊序列,随后引发RNA聚合酶II的催化作用。转录起始蛋白主要包含两种: TBP ( TATA-binding protein )和TLP ( TBP-like protein )。已经证明前者是一种能与TATA盒结合的蛋白,TATA盒是位于基因上游并带有TATA重复结构的一段序列。有时候,如果其他一些启动子缺乏TATA盒结构(TATA less)那么TLP就会参与其转录,但是关于TLP在启动子区的识别位点还所知甚少。本实验主要包括两个方面:1、TBP和TLP蛋白是否能与hTERT基因启动子区结合。2、TBP和TLP蛋白是否可以调节hTERT基因启动子的表达及所涉及的细胞生物学活动。主要的实验方法及结果如下:1.构建含有TBP基因和TLP基因的原核表达载体pEK-TBP和pEK-TLP,转化大肠杆菌E. coli诱导表达原核6×His-TBP和6×His-TLP融合蛋白,经SDS-PAGE鉴定、变性调节下利用亲和层析纯化和透析复性。2. EMSA试验证实TBP蛋白可以与hTERT基因启动子核心区特异性结合,但TLP蛋白不与hTERT基因启动子区结合。3.荧光素酶报告基因分析观察TBP对hTERT启动子区的调节。构建含有TBP基因的真核表达载体,与含有hTERT启动子的荧光酶报告载体共转染HeLa细胞,荧光酶活性分析显示TBP蛋白可以激活hTERT启动子的表达。4.染色体免疫共沉淀(ChIP)检测到TBP蛋白在HeLa细胞内可以与hTERT启动子结合。5.使用Footprinting分析TBP蛋白与hTERT启动子结合位点的序列。结论:TBP蛋白可以与hTERT基因启动子核心区特异性结合,但TLP蛋白不与hTERT基因启动子区结合。TBP蛋白在细胞内也可与hTERT启动子结合并激活其表达。TBP很可能是一个重要的转录调节因子并参与hTERT的激活。目的:基因启动子的甲基化是肿瘤形成过程中的一个重要的机制。一些基因启动子在宫颈癌中高甲基化已经有了相关报道。但是,关于宫颈癌前病变的甲基化还所知甚少。为了获得更多关于宫颈癌形成过程中甲基化进程的知识,并筛选和调整一个能用于诊断宫颈刮片标本中宫颈癌和鳞状上皮内高度病变的诊断标志物,我们利用实时定量甲基特异性PCR(QMSP)分析了九个基因启动子的甲基化状态,这些基因在宫颈癌中高甲基化已经有过相关报道。方法:石蜡包埋正常宫颈组织(20例),鳞状上皮内低度病变(20例),鳞状上皮内高度病变(20例),腺癌(20例)和鳞癌(40例)。对应的宫颈刮片标本55例。提取DNA后,用QMSP方法分析DAPK, CALCA, ESR1, APC, RAR-β2, TFPI2, SPARC, CCNA1和CADM1基因的甲基化状态。结果:所有基因的甲基化水平都随着标本的恶性度增加而增加。除了CALCA, ESR1和RAR-β2基因,其他基因启动子甲基化的样品数量也随着标本的恶性度增加而增加。DAPK和CADM1基因在HSIL标本中就已经显著的甲基化了,但CCNA1, TFPI2和ESR1基因主要在宫颈癌中甲基化。宫颈刮片标本中甲基化比率反映了病变的甲基化状态。此外,CCNA1基因在宫颈癌和HSIL病例中甲基化比例很高,分别为80%和56%,而LSIL中只有25%甲基化。结论:无论在石蜡包埋还是宫刮片标本中,标本甲基化的数目和水平都在肿瘤发生形成过程中增加。DAPK和CADM1基因是最好的区分LSIL和HSIL病变的基因启动子,这对SIL病变的治疗可能很重要。更多还原

【Abstract】 Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase in human, has been identified the rate-limiting factor in telomerase activity, but its detailed mechanism of transcription regulation remains unclear. Many investigations are focusing on the precise mechanism of hTERT transcription regulation. The transcription of genes encoding functional proteins in cells has been identified to be dependent on activity of RNA polymerase II. However, RNA polymerase II does not directly bind to promoter as elucidated so far but mediated by cellular proteins. The proteins for initiating transcription include two major proteins: TBP (TATA-binding protein) and TLP (TBP-like protein). In this investigation, TBP and TLP were expressed and purificated. The interaction of TBP with hTERT promoter was identified, but the interaction of TLP with hTERT promoter was not identified. The in vivo interaction between TBP and hTERT promoter was identified by chromatin immunoprecipitation (ChIP) assay. Transfection of TBP into HeLa cells presented marked transactivation of hTERT promoter. These results suggested that TBP protein might be a transcription regulator and take part in activation of hTERT. Objectives: Gene promoter hypermethylation is an important mechanism involved in carcinogenesis. Several gene promoters are known to be hypermethylated in cervical cancer. However, not much is known about hypermethylation in cervical cancer precursor lesions. In order to obtain more insight in the course of hypermethylation throughout cervical carcinogenesis, and to allow selection and adjustment of a marker panel for the detection of cervical cancer and high grade (H)SIL in scrapings, the hypermethylation status of nine gene promoters, previously reported to be hypermethylated in cervical cancer, was analyzed using quantitative methylation specific PCR (QMSP).Methods: Paraffin embedded tissue from normal cervix (n=20), LSIL (n=20), HSIL (n=20), adenocarcinomas (AC) (n=20) and squamous cell cervical cancers (SCC) (n=40) was retrieved together with 55 corresponding cervical scrapings and DNA was extracted. Promoter methylation analysis was performed using QMSP for DAPK, CALCA, ESR1, APC, RAR-β2, TFPI2, SPARC, CCNA1 and CADM1.Results: The level of methylation of all gene promoters was increased with severity of the underlying lesion. The number of samples methylated of most gene promoters was increased as well with the severity of the lesion, except for CALCA, ESR1 and RAR-β2. DAPK and CADM1 became more prominently methylated in HSIL lesions compared to LSIL, while CCNA1, TFPI2 and ESR1 became mainly methylated in cervical cancers. Methylation ratios determined in the scrapings were reflecting the methylation status of the underlying lesion. Furthermore, in cervical scrapings, CCNA1 was most frequently hypermethylated in cervical cancer cases (80%) and in HSIL (56%), while only 25% of LSIL showed hypermethylation.Conclusion: The number of samples methylated and the level of methylation was increased during carcinogenesis in paraffin embedded tissue as well as in corresponding cervical scrapings. DAPK and CADM1 gene promoter methylation were the best gene promoters to distinguish LSIL vs HSIL lesions, which might be important for treatment of SIL lesions.更多还原