人PNRC基因转录调控的研究

发布时间：2018-01-11 21:07

本文关键词：人PNRC基因转录调控的研究　出处：《第三军医大学》2008年博士论文　论文类型：学位论文

【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。
[Abstract]:Proline rich nuclear receptor coactivator (proline-rich nuclear receptor coactivator, PNRC) is a kind of new nuclear receptor recently cloned and identified the coactivator, is a growth factor in steroid derived (steridogentic factor 1, SF1) as bait, using yeast two hybrid system was obtained through.PNRC library screening in C SH3 from human breast cDNA (SRC homology 3) binding motif play nuclear receptor coactivator, in a ligand dependent manner with a variety of nuclear receptor interactions, but also with the orphan receptor SF1 and ERR alpha 1 in a ligand independent manner in.PNRC interaction as a nuclear receptor coactivator outside also, through the SH3 binding motif of growth factors that regulate the /Ras signaling pathway, the expression of activated.PNRC competitive inhibition of Ras protein in tumor tissues and cells is significantly lower than that of normal tissues and cells, the It is suggested that the transcriptional inhibition of PNRC is related to the development of a variety of tumors.
At present, the transcriptional regulation mechanism of PNRC is not very clear. In order to study the transcriptional regulation mechanism of PNRC gene and its correlation with the occurrence and development of many kinds of tumors, we carried out the following research.
Determination of the transcriptional starting site of the PNRC gene in 1. people and the activity analysis of the promoter
Using rapid amplification of 5 'cDNA (rapid amplification of cDNA ends terminal, RACE) to determine the PNRC transcription start site base for G, the transcription initiation site was reported by PNRC mRNA (NM 006813) the 5' end of the first base ahead of the 27 bp. predicted by bioinformatics software (http://www.fruitfly.org/ cgi-bin/seq_tools/promoter.pl.) sequence of the human PNRC gene 5 'flanking region about 2100bp analysis, forecast 5' flanking region has the function of promoter region. The application of TransFac professional 8.1 (http://jupiter.coh.org/ cgi-bin/ transfac/bin/start.cgi) software to analyze the region, obtain the possible transcription factor binding sites. The luciferase reporter plasmid construct containing the PNRC gene 5 'upstream sequence, using liposome transfection, luciferase activity was detected in the promoter region of PNRC were analyzed. Results showed that, The minimum promoter activity is located in -123 ~ +27. The prediction results show that there may be a binding site of NFY and RFX1 transcription factors in this region.
The binding of NFY and RFX1 to the active region of the PNRC gene promoter in 2. cells
Chromatin immunoprecipitation assay (ChIP) and electrophoretic mobility shift assay (electrophoresis mobility shift and supershift assay, EMSA) proved that both the cells and the extracellular domain were combined with the promoter region.
3.NFY and RFX1 on PNRC gene promoter activity and regulation of NFY and RFX1 eukaryotic expression plasmid and the recombinant plasmid PNRC promoter reporter gene or mutation reporter gene plasmid (including transcription factor binding site mutation) that CO transfection experiments: NFY and RFX1 with PNRC promoter -123 ~ +27 binding region, the inhibition of PNRC promoter the activity of RT-PCR and Western blot showed that overexpression of NFY and RFX1 expression of PNRC in HepG2 cells.
Study on the methylation status of human PNRC gene promoter CpG island in 4. breast cancer cells
The use of "on the promoter region of PNRC was analyzed by MethPrimer software, CpG Island, PCR (Bisulfite sequencing sequencing by sulfide PCR, BSP), the methylation status of PNRC, detection of CpG island in the promoter region showed that methylation of PNRC gene in breast cancer cells existed in the promoter region of CpG island.
Regulation of PNRC transcription by CpG Island demethylation of 5.PNRC gene promoter
PNRC containing promoter reporter plasmids were transfected into breast cancer cells, cells with 5- methyltransferase inhibitor aza -2 '- deoxycytidine (5-Aza-2 -deoxycytidine, 5-Aza-CdR) after treatment, detection of PNRC gene promoter activity; RT-PCR, Northern hybridization, detection of breast cancer cells Western Blot expression by PNRC treatment after 5-Aza-CdR. The results showed that: PNRC gene promoter CpG island methylation can enhance PNRC promoter activity and expression of PNRC.
The study of sequence flanking region of human PNRC gene 5 'were analyzed a series of deletions, determine the promoter activity of -123 ~ +27 minimum area, and identified with the combination of transcription factors NFY and RFX1 on the regulation of PNRC promoter. In addition, we also studied from the genetic regulation of methylation status PNRC gene promoter CpG Island epigenetic, found PNRC methylation of MCF-7 gene in breast cancer cells existed in the promoter region of CpG Island, the PNRC gene promoter CpG island methylation can enhance PNRC promoter activity and expression of PNRC, further proved that the transcription of PNRC in breast cancer cell inhibition methyl PNRC and promoter CpG island. The results of this study laid the foundation for the correlation between the elucidation of PNRC gene transcriptional regulation mechanism of PNRC and its transcription inhibition and tumor.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346;R730.2

【引证文献】