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  本文關(guān)鍵詞：人PNRC基因轉(zhuǎn)錄調(diào)控的研究　出處：《第三軍醫(yī)大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 富含脯氨酸的核受體輔活化因子 啟動子分析 核因子Y 調(diào)節(jié)因子X1 轉(zhuǎn)錄調(diào)節(jié) 甲基化

【摘要】： 富含脯氨酸的核受體輔活化因子(proline-rich nuclear receptor coactivator, PNRC)是最近克隆和鑒定的一種新的核受體輔活化因子,是以類固醇源性生長因子(steridogentic factor 1, SF1)作誘餌,利用酵母雙雜交系統(tǒng)從人乳腺cDNA表達(dá)文庫中篩選得到的。PNRC通過其位于C端的SH3(src homology 3)結(jié)合模體發(fā)揮核受體輔活化因子作用,能以配體依賴的方式與多種核受體相互作用,還能與孤兒受體SF1和ERRα1以配體非依賴的方式相互作用。PNRC除了作為核受體的輔活化因子外,還通過SH3結(jié)合模體調(diào)節(jié)生長因子/Ras信號通路,競爭性抑制Ras蛋白的激活。PNRC在多種腫瘤組織及細(xì)胞中的表達(dá)明顯低于正常組織和細(xì)胞,這提示PNRC的轉(zhuǎn)錄抑制與多種腫瘤的發(fā)生、發(fā)展相關(guān)。 目前關(guān)于PNRC的轉(zhuǎn)錄調(diào)控機(jī)制尚不十分清楚,為了研究PNRC基因的轉(zhuǎn)錄調(diào)控機(jī)制及其轉(zhuǎn)錄抑制與多種腫瘤的發(fā)生、發(fā)展的相關(guān)性,我們進(jìn)行了如下的研究: 1.人PNRC基因轉(zhuǎn)錄起始位點(diǎn)的確定及啟動子的活性分析 采用5′端cDNA末端快速擴(kuò)增法(rapid amplification of cDNA ends, RACE)確定了PNRC轉(zhuǎn)錄起始位點(diǎn)的堿基為G,該轉(zhuǎn)錄起始位點(diǎn)較報道的PNRC mRNA (NM 006813) 5′端第一個堿基提前了27 bp。通過生物信息學(xué)預(yù)測軟件(http://www.fruitfly.org/ cgi-bin/seq_tools/promoter.pl.)對人PNRC基因的5′側(cè)翼區(qū)約2100bp的序列進(jìn)行分析,預(yù)測5′側(cè)翼區(qū)內(nèi)具有啟動子功能的區(qū)域。應(yīng)用TransFac professional 8.1(http://jupiter.coh.org/ cgi-bin/transfac/bin/start.cgi)軟件對該區(qū)域進(jìn)行分析,獲得可能的轉(zhuǎn)錄因子結(jié)合位點(diǎn)。通過構(gòu)建包含PNRC基因5′上游序列的熒光素酶報告質(zhì)粒,利用脂質(zhì)體瞬時轉(zhuǎn)染,檢測熒光素酶活性,對PNRC啟動子區(qū)進(jìn)行分析,結(jié)果顯示,啟動子活性最小區(qū)域位于-123 ~ +27,預(yù)測結(jié)果表明,在這一區(qū)域可能存在著NFY和RFX1轉(zhuǎn)錄因子結(jié)合位點(diǎn)。 2.細(xì)胞內(nèi)、細(xì)胞外鑒定NFY和RFX1與PNRC基因啟動子活性區(qū)域的結(jié)合 染色質(zhì)免疫沉淀(chromatin immunoprecipitation assay, ChIP)及電泳遷移率變動實(shí)驗(electrophoresis mobility shift and supershift assay, EMSA)證明:NFY和RFX1可在細(xì)胞內(nèi)、細(xì)胞外與PNRC啟動子區(qū)域相結(jié)合。 3.NFY和RFX1對PNRC基因啟動子活性的調(diào)控NFY和RFX1真核表達(dá)質(zhì)粒與PNRC啟動子重組報告基因質(zhì)�；蚺c突變報告基因質(zhì)粒(含轉(zhuǎn)錄因子結(jié)合位點(diǎn)突變)的共轉(zhuǎn)染實(shí)驗表明:NFY和RFX1通過與PNRC啟動子-123 ~ +27區(qū)域結(jié)合,抑制PNRC啟動子的活性,RT-PCR和Western blot證明:過表達(dá)NFY和RFX1可下調(diào)HepG2細(xì)胞中PNRC的表達(dá)。 4.乳腺癌細(xì)胞中人PNRC基因啟動子CpG島甲基化狀態(tài)的研究 運(yùn)用“MethPrimer”軟件對PNRC啟動子區(qū)進(jìn)行分析,預(yù)測CpG島,通過硫化測序PCR (Bisulfite sequencing PCR, BSP),檢測PNRC啟動子區(qū)CpG島的甲基化狀況,結(jié)果顯示:乳腺癌細(xì)胞中PNRC基因啟動子區(qū)存在CpG島的甲基化。 5.PNRC基因啟動子CpG島去甲基化對PNRC轉(zhuǎn)錄的調(diào)節(jié) 將含PNRC啟動子序列的報告質(zhì)粒轉(zhuǎn)染乳腺癌細(xì)胞,細(xì)胞再經(jīng)甲基轉(zhuǎn)移酶抑制劑5-氮雜-2’-脫氧胞苷(5-aza-2’-deoxycytidine, 5-Aza-CdR)處理后,檢測PNRC基因啟動子的活性;RT-PCR、Northern雜交、Western Blot檢測乳腺癌細(xì)胞經(jīng)5-Aza-CdR處理后PNRC的表達(dá)情況。結(jié)果證明:PNRC基因啟動子CpG島去甲基化可增強(qiáng)PNRC啟動子活性并上調(diào)PNRC的表達(dá)。 本研究對人PNRC基因5′側(cè)翼區(qū)序列進(jìn)行了一系列缺失分析,確定了啟動子的最小活性區(qū)域-123 ~ +27,并鑒定了與之相結(jié)合的轉(zhuǎn)錄因子NFY和RFX1對PNRC啟動子的調(diào)節(jié)作用。此外,我們還從表觀遺傳學(xué)調(diào)控方面研究了PNRC基因啟動子CpG島的甲基化狀態(tài),發(fā)現(xiàn)乳腺癌細(xì)胞MCF-7中PNRC基因啟動子區(qū)存在CpG島的甲基化,對PNRC基因啟動子CpG島的去甲基化可增強(qiáng)PNRC啟動子活性并上調(diào)PNRC的表達(dá),進(jìn)一步證明了PNRC在乳腺癌細(xì)胞中的轉(zhuǎn)錄抑制與PNRC啟動子CpG島的甲基化相關(guān)。本研究結(jié)果為闡明PNRC基因自身轉(zhuǎn)錄調(diào)控機(jī)制以及PNRC的轉(zhuǎn)錄抑制與腫瘤的相關(guān)性奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R346;R730.2

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 彭旭;ITGB4BP調(diào)控TGF-β1表達(dá)的實(shí)驗研究[D];第三軍醫(yī)大學(xué);2011年

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本文編號：1411208