【作者】 张艳；

【导师】 周度金；

【作者基本信息】 第三军医大学 ， 免疫学， 2008， 博士

【摘要】 富含脯氨酸的核受体辅活化因子(proline-rich nuclear receptor coactivator, PNRC)是最近克隆和鉴定的一种新的核受体辅活化因子,是以类固醇源性生长因子(steridogentic factor 1, SF1)作诱饵,利用酵母双杂交系统从人乳腺cDNA表达文库中筛选得到的。PNRC通过其位于C端的SH3(src homology 3)结合模体发挥核受体辅活化因子作用,能以配体依赖的方式与多种核受体相互作用,还能与孤儿受体SF1和ERRα1以配体非依赖的方式相互作用。PNRC除了作为核受体的辅活化因子外,还通过SH3结合模体调节生长因子/Ras信号通路,竞争性抑制Ras蛋白的激活。PNRC在多种肿瘤组织及细胞中的表达明显低于正常组织和细胞,这提示PNRC的转录抑制与多种肿瘤的发生、发展相关。目前关于PNRC的转录调控机制尚不十分清楚,为了研究PNRC基因的转录调控机制及其转录抑制与多种肿瘤的发生、发展的相关性,我们进行了如下的研究:1.人PNRC基因转录起始位点的确定及启动子的活性分析采用5′端cDNA末端快速扩增法(rapid amplification of cDNA ends, RACE)确定了PNRC转录起始位点的碱基为G,该转录起始位点较报道的PNRC mRNA (NM 006813) 5′端第一个碱基提前了27 bp。通过生物信息学预测软件(http://www.fruitfly.org/ cgi-bin/seq_tools/promoter.pl.)对人PNRC基因的5′侧翼区约2100bp的序列进行分析,预测5′侧翼区内具有启动子功能的区域。应用TransFac professional 8.1(http://jupiter.coh.org/ cgi-bin/transfac/bin/start.cgi)软件对该区域进行分析,获得可能的转录因子结合位点。通过构建包含PNRC基因5′上游序列的荧光素酶报告质粒,利用脂质体瞬时转染,检测荧光素酶活性,对PNRC启动子区进行分析,结果显示,启动子活性最小区域位于-123 ~ +27,预测结果表明,在这一区域可能存在着NFY和RFX1转录因子结合位点。2.细胞内、细胞外鉴定NFY和RFX1与PNRC基因启动子活性区域的结合染色质免疫沉淀(chromatin immunoprecipitation assay, ChIP)及电泳迁移率变动实验(electrophoresis mobility shift and supershift assay, EMSA)证明:NFY和RFX1可在细胞内、细胞外与PNRC启动子区域相结合。3.NFY和RFX1对PNRC基因启动子活性的调控NFY和RFX1真核表达质粒与PNRC启动子重组报告基因质粒或与突变报告基因质粒(含转录因子结合位点突变)的共转染实验表明:NFY和RFX1通过与PNRC启动子-123 ~ +27区域结合,抑制PNRC启动子的活性,RT-PCR和Western blot证明:过表达NFY和RFX1可下调HepG2细胞中PNRC的表达。4.乳腺癌细胞中人PNRC基因启动子CpG岛甲基化状态的研究运用“MethPrimer”软件对PNRC启动子区进行分析,预测CpG岛,通过硫化测序PCR (Bisulfite sequencing PCR, BSP),检测PNRC启动子区CpG岛的甲基化状况,结果显示:乳腺癌细胞中PNRC基因启动子区存在CpG岛的甲基化。5.PNRC基因启动子CpG岛去甲基化对PNRC转录的调节将含PNRC启动子序列的报告质粒转染乳腺癌细胞,细胞再经甲基转移酶抑制剂5-氮杂-2’-脱氧胞苷(5-aza-2’-deoxycytidine, 5-Aza-CdR)处理后,检测PNRC基因启动子的活性;RT-PCR、Northern杂交、Western Blot检测乳腺癌细胞经5-Aza-CdR处理后PNRC的表达情况。结果证明:PNRC基因启动子CpG岛去甲基化可增强PNRC启动子活性并上调PNRC的表达。本研究对人PNRC基因5′侧翼区序列进行了一系列缺失分析,确定了启动子的最小活性区域-123 ~ +27,并鉴定了与之相结合的转录因子NFY和RFX1对PNRC启动子的调节作用。此外,我们还从表观遗传学调控方面研究了PNRC基因启动子CpG岛的甲基化状态,发现乳腺癌细胞MCF-7中PNRC基因启动子区存在CpG岛的甲基化,对PNRC基因启动子CpG岛的去甲基化可增强PNRC启动子活性并上调PNRC的表达,进一步证明了PNRC在乳腺癌细胞中的转录抑制与PNRC启动子CpG岛的甲基化相关。本研究结果为阐明PNRC基因自身转录调控机制以及PNRC的转录抑制与肿瘤的相关性奠定了基础。更多还原

【Abstract】 PNRC (Proline-rich Nuclear Receptor Coactivator) was previously identified using bovine SF-1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC was found to interact with the ligand-binding domains of all the nuclear receptors tested, including ERα, ERβ, PR, GR, TR, RAR, and RXR, in a ligand-dependent manner. PNRC was also found to interact in a ligand-independent manner with orphan receptors such as SF1 and ERRα. Unlike most of the coactivators that interact with nuclear receptors through their LXXLL motif, this new coactivator interacts with nuclear receptors through a proline-rich Src homology domain-3 (SH3)-binding motif, S-D (E)-P-P-S-P-S. In addition to functioning as a nuclear receptor coactivator, PNRC also plays a role in the growth factor/Ras-signaling pathway through its interaction with Grb2, a central adaptor protein in Ras pathway. Significantly reduced PNRC mRNA expression in stomach, colorectal, and hepatocellular carcinomas and in breast cancer was observed in comparison with the corresponding non-cancerous tissues. In addition, time-dependent induction of mRNA expression of PNRC was observed during 5-azacytidine treatment. These data suggest a possibility of PNRC as a tumor-related gene and that the regulation of PNRC expression plays a role in human multistage carcinogenesis. However, very little is known about the regulation of the expression of PNRC gene itself. To better understand the molecular mechanisms that regulate the expression of PNRC gene, we carried out this study and the major results are summarized below:1. The transcriptional initiation site and the minimal promoter region of the human PNRC gene were identified in this study. Using 5’RACE approach, we identified a major transcriptional initiation site at a nucleotide G, 27 nt more 5’upstream from the 5’end of previous published longest PNRC cDNAs (NM 006813). The sequences in the 2100 bp 5′flanking region from this transcriptional start site of PNRC gene was then analyzed for promoter prediction (http://www.fruitfly.org/cgi-bin/seq_tools/promoter.pl) and for putative transcriptional factor binding site analyses (TRANSFAC Professional 8.1 software in the Biobase Biological Database) (www. biobase.de/pages/products/databases. Htm l # transfac). To identify the promoter sequences of the human PNRC gene, a series of luciferase reporter constructs containing various 5′flanking region deletions of PNRC gene were generated by PCR and they were transient transfected into HepG2 cell by Liposome. Functional assays showed that the minimal promoter region of the human PNRC gene was mapped in the region from nucleotides -123 to +27, and the predicted transcription factor binding sites of NFY and RFX1 were located in this region.2. Chromatin immunoprecipitation (ChIP) analysis and electrophoretic mobility shift assay (EMSA) revealed that transcription factor NFY and RFX1 interacted with the–123 ~ +27 promoter region of the human PNRC gene in vivo and in vitro.3. The results of the co-transfection experiments showed that the transcriptional activity of human PNRC promoter was found to be regulated negatively by transcription factors NFY and RFX1, through binding two NFY putative binding sites and the RFX1-binding sequence within PNRC promoter region. RT-PCR and Western blot further demonstrated that overexpression of NFY or RFX1 negatively regulated PNRC expression in HepG2 cells.4. After prediction of CpG island in promoter of PNRC gene using“MethPrimer”software, the status of methylation of CpG island in promoter of PNRC gene was analyzed using bisulfite sequencing PCR (BSP) and sequence assay. The results revealed that the CpG island in promoter of PNRC was methylated only in breast cancer MCF-7 cells but not in normal breast MCF-10A cells.5. MCF-7 cells transfected with the luciferase report plasmid that contains the promoter sequence of PNRC were treated with methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-Aza-CdR) and the promoter activity of PNRC was detected by luciferase assay. The expression of PNRC was analyzed by RT-PCR, Northern hybridization and Western blot after MCF-7 cells were treated with 5-Aza-CdR. The results showed that the promoter activity of PNRC in MCF-7 cells was levated upon the treatment of 5-Aza-CdR, and the levels of mRNA and protein of PNRC in MCF-7 cells were increased dramatically in response to the treatment of 5-Aza- CdR.In summary, in this study we have cloned and characterized the 5′flanking region of the human PNRC gene. Potential transcriptional start sites were determined by 5′RACE analysis. The minimal promoter region of the human PNRC gene was mapped in the region from nucleotides -123 to +27. The transcriptional activity of human PNRC promoter was found to be regulated negatively by transcription factors NFY and RFX1, through binding two NFY putative binding sites and the RFX1-binding sequence within PNRC promoter region. In addition, we also investigated the epigenetic regulation of PNRC gene. The results from this study suggest that hypermethylation of CpG island in promoter of PNRC is probably responsible for PNRC expression silencing in breast cancer cells, and the methyltransferase inhibitor such as 5-Aza-dc can effectively activate the expression of PNRC. The information generated from this study provides a solid base for further study of the transcriptional regulation of PNRC gene and the development of a novel strategy in breast cancer diagnosis and treatment by detecting and modulating the DNA methylation of PNRC gene.更多还原