本文選題：NFBD1 + 啟動子��； 參考：《重慶醫(yī)科大學(xué)》2007年博士論文

【摘要】： NFBD1(Nuclear Factor with BRCT Domains Protein 1),也稱MDC1(Mediator of DNA Damage Checkpoint Protein 1),是一個參與細胞內(nèi)DNA損傷后細胞應(yīng)答反應(yīng)的重要分子。我們研究小組在前期曾發(fā)現(xiàn)NFBD1在DNA損傷后出現(xiàn)轉(zhuǎn)錄下調(diào)并通過和P53互作而調(diào)節(jié)細胞凋亡,但NFBD1的轉(zhuǎn)錄調(diào)控機制仍不清楚,同時試驗結(jié)果也提示NFBD1很可能參與細胞生長凋亡等的調(diào)節(jié)。本論文即在此基礎(chǔ)上,對NFBD1的轉(zhuǎn)錄調(diào)控及其調(diào)節(jié)細胞生長凋亡的功能進行了相對系統(tǒng)的研究。分述如下: (1)NFBD1啟動子的克隆、鑒定與初步分析:首先應(yīng)用5' RACE技術(shù)結(jié)合生物信息學(xué)分析鑒定了NFBD1的轉(zhuǎn)錄起始位點,首次發(fā)現(xiàn)NFBD1至少存在4種豐度和轉(zhuǎn)錄起始位點不同的轉(zhuǎn)錄剪接變異體。然后采用PCR定向克隆和酶切亞克隆策略,構(gòu)建了覆蓋NFBD1基因5'側(cè)翼區(qū)起始密碼子ATG上游5 kb區(qū)域的一系列NFBD1啟動子熒光素酶報告基因重組體。啟動子活性分析表明,NFBD1主要啟動子區(qū)域定位于其主要轉(zhuǎn)錄起始位點區(qū)域附近1.5 kb的區(qū)域內(nèi),同時在其上游還發(fā)現(xiàn)了一個活性較低的啟動子。采用轉(zhuǎn)錄因子結(jié)合位點預(yù)測分析軟件分析表明,NFBD1啟動子缺乏TATA盒,但含有典型的CCAAT盒和GC盒以及其它潛在的轉(zhuǎn)錄因子結(jié)合位點,提示Sp1和NF-Y等轉(zhuǎn)錄因子可能參與NFBD1的轉(zhuǎn)錄調(diào)控。 (2)NFBD1核心啟動子區(qū)域的鑒定與分析:針對已經(jīng)初步鑒定的NFBD1主要啟動子區(qū)域,進一步采用限制性酶切聯(lián)合DNA blunting方法構(gòu)建NFBD1啟動子熒光素酶報告基因系列刪除體。啟動子活性分析表明,NFBD1核心啟動子區(qū)域定位于其主要轉(zhuǎn)錄起始位點區(qū)域附近325 bp的區(qū)域內(nèi),該區(qū)域分別包含有兩個高度保守的Sp1和NF-Y結(jié)合位點,但沒有發(fā)現(xiàn)保守的STAT1結(jié)合位點。ChIP和EMSA實驗結(jié)果表明,Sp1和NF-Y可以與NFBD1啟動子在體外(in vitro)和細胞內(nèi)(in vivo)結(jié)合,而STAT1則不與該NFBD1啟動子結(jié)合。定點突變分析實驗結(jié)果表明,NFBD1核心啟動子區(qū)域內(nèi)NF-Y結(jié)合位點的突變對NFBD1啟動子活性沒有明顯影響,但Sp1結(jié)合位點的突變導(dǎo)致NFBD1啟動子活性的消失,提示Sp1可能在NFBD1的轉(zhuǎn)錄調(diào)控中起著更為重要的作用。 (3)NFBD1的DNA損傷后轉(zhuǎn)錄下調(diào)機制研究:采用阿霉素處理A549或HeLa細胞建立DNA損傷細胞模型,發(fā)現(xiàn)阿霉素處理后細胞內(nèi)NFBD1 mRNA和蛋白質(zhì)水平顯著降低,而Sp1的表達水平?jīng)]有明顯變化。磷酸酶處理實驗表明,阿霉素處理后Sp1出現(xiàn)磷酸化修飾,同時ChIP實驗表明,阿霉素處理導(dǎo)致Sp1與NFBD1啟動子的結(jié)合活性降低。NFBD1啟動子熒光素酶報告基因系列刪除體分析實驗表明,Sp1參與DNA損傷后的NFBD1轉(zhuǎn)錄下調(diào)。Sp1的特異性抑制劑光輝霉素A處理和siRNA介導(dǎo)的Sp1表達抑制導(dǎo)致NFBD1啟動子活性和內(nèi)源性NFBD1 mRNA和蛋白質(zhì)水平的降低,同時,siRNA介導(dǎo)的Sp1表達抑制也提高了細胞對化療藥物阿霉素的敏感性。這些結(jié)果表明,Sp1介導(dǎo)DNA損傷后NFBD1的轉(zhuǎn)錄下調(diào),這在DNA損傷應(yīng)答反應(yīng)中起著重要的作用。 (4)STAT1在NFBD1轉(zhuǎn)錄調(diào)控中的作用研究:半定量RT-PCR實驗結(jié)果表明,DNA損傷后NFBD1出現(xiàn)明顯的轉(zhuǎn)錄下調(diào),STAT1 mRNA水平明顯升高,其相應(yīng)靶基因也同時出現(xiàn)轉(zhuǎn)錄激活或抑制。但蛋白質(zhì)印跡分析實驗結(jié)果表明,DNA損傷后STAT1蛋白質(zhì)水平僅呈現(xiàn)輕微升高,且STAT1的磷酸化修飾和轉(zhuǎn)位/移位情況沒有明顯變化。雖然STAT1抑制劑氟達拉濱處理非特異性地顯著降低了NFBD1的啟動子活性和mRNA水平,但IFNγ處理誘發(fā)的內(nèi)源性STAT1激活和siRNA介導(dǎo)的STAT1表達抑制對NFBD1的啟動子活性和mRNA水平?jīng)]有明顯影響。這些結(jié)果表明,STAT1不參與NFBD1的直接轉(zhuǎn)錄調(diào)控。 (5)NFBD1在細胞生長、凋亡中的作用研究:半定量RT-PCR和蛋白質(zhì)印跡分析實驗結(jié)果表明,篩選到的短鏈NFBD1 siRNA可以成功抑制內(nèi)源NFBD1的表達,抑制率幾乎100%。對HeLa等細胞進行NFBD1 siRNA瞬時轉(zhuǎn)染,發(fā)現(xiàn)NFBD1 siRNA可以顯著抑制細胞生長。FACS分析和蛋白質(zhì)印跡分析結(jié)果表明,NFBD1 siRNA可以導(dǎo)致細胞內(nèi)DNA損傷的累積、G2/M期檢測點的激活和G2/M期停滯。采用細胞同步化技術(shù),制備不同細胞周期時相的樣品,進行NFBD1的基因表達譜分析,發(fā)現(xiàn)NFBD1在G2/M期表達水平升高,在G1、S期表達水平降低,表明NFBD1為一G2/M期基因,并參與細胞周期進程或調(diào)控。FACS分析結(jié)果表明,NFBD1 siRNA瞬時轉(zhuǎn)染導(dǎo)致sub-G1峰的出現(xiàn),同時蛋白質(zhì)印跡分析觀察到了Caspase 3和PARP的剪接激活,表明NFBD1 siRNA誘發(fā)了細胞凋亡。另外蛋白質(zhì)印跡分析結(jié)果還表明,該凋亡過程中P53及其下游靶分子bax和puma也沒有明顯變化,但noxa在mRNA和蛋白質(zhì)水平上均顯著升高,強烈提示P53非依賴性的Noxa轉(zhuǎn)錄激活可能在NFBD1 siRNA誘發(fā)細胞凋亡的過程中起著重要作用。另外,MTT分析結(jié)果也表明,NFBD1 siRNA顯著提高了細胞對阿霉素、順鉑等化療藥物的敏感性。這些結(jié)果表明,作為一新的G2/M期基因,NFBD1不僅參與正常的細胞生長,而且參與調(diào)節(jié)細胞凋亡過程;NFBD1也是一個有效的放化療增敏劑藥物和基因治療藥物靶點。 本論文對NFBD1轉(zhuǎn)錄調(diào)控及其調(diào)節(jié)細胞生長凋亡的分子功能的系統(tǒng)研究進一步加深了我們對NFBD1分子功能和分子行為機制的認(rèn)識,將為進一步深入開展NFBD1的基礎(chǔ)理論研究、以及促進以NFBD1為分子靶點的抗癌藥物開發(fā)研究均具有積極的理論和現(xiàn)實意義。
[Abstract]:NFBD1 ( Nuclear Factor with BRCT Domains Protein 1 ) , also known as MDC1 ( Proliferation of DNA Damage Checkpoint Protein 1 ) , is an important molecule involved in the cellular response after DNA damage in cells . Our research team has found that NFBD1 regulates apoptosis after DNA damage , but also suggests that NFBD1 may participate in regulation of cell growth and apoptosis .






( 1 ) Cloning , identification and preliminary analysis of NFBD1 promoter : First , the transcription initiation site of NFBD1 was identified by using 5 ' RACE technique and bioinformatics analysis .






( 2 ) Identification and analysis of NFBD1 core promoter region : A series of deletion bodies of NFBD1 promoter luciferase reporter gene were constructed by restriction enzyme digestion combined with DNA blunting method . The promoter activity analysis showed that the NFBD1 promoter region was located in the region of 325 bp near its main transcription initiation site , but the conserved STAT1 binding site was not found . The results of site - directed mutation assay showed that the mutation of NF - Y binding site in NFBD1 promoter region did not have a significant effect on the activity of NFBD1 promoter , but the mutation of Sp1 binding site led to the disappearance of NFBD1 promoter activity , suggesting that Sp1 might play a more important role in the transcriptional regulation of NFBD1 .






( 3 ) Down - regulation mechanism of NFBD1 after DNA damage : A DNA damage cell model was established by adriamycin - treated A549 or HeLa cells . The expression level of NFBD1 mRNA and protein was significantly decreased after adriamycin treatment . The results showed that Sp1 induced phosphorylation modification of NFBD1 after DNA damage . The results showed that the transcription of NFBD1 was regulated by the expression of the specific inhibitor of Sp1 . These results suggested that Sp1 mediated down - regulation of NFBD1 after DNA damage , which played an important role in DNA damage response .






( 4 ) STAT1 plays a role in the regulation of NFBD1 transcription . The results of semi - quantitative RT - PCR show that the expression of STAT1 mRNA is down - regulated after DNA damage , and the transcription activation or inhibition of STAT1 mRNA appears at the same time . However , the experimental results of Western blot analysis showed that STAT1 protein level was only slightly increased after DNA damage , but the phosphorylation modification and mRNA level of STAT1 did not significantly affect the promoter activity and mRNA level of NFBD1 . These results suggest that STAT1 is not involved in the direct transcription regulation of NFBD1 .






The results of FACS analysis and Western blot analysis showed that NFBD1 siRNA could significantly inhibit the expression of NFBD1 siRNA . The results of FACS analysis and Western blot showed that NFBD1 siRNA could significantly inhibit the cell growth .






This paper further deepened our understanding of NFBD1 molecule function and molecular behavior mechanism , and further deepened our understanding of NFBD1 ' s molecular function and molecular behavior mechanism , and will provide theoretical and practical significance to further study the basic theory of NFBD1 and to promote the development of anti - cancer drugs with NFBD1 as molecular target .

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R346

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