NFBD1启动子克

发布时间：2018-04-11 12:18

本文选题：NFBD1 + 启动子　；参考：《重庆医科大学》2007年博士论文

【摘要】： NFBD1(Nuclear Factor with BRCT Domains Protein 1),也称MDC1(Mediator of DNA Damage Checkpoint Protein 1),是一个参与细胞内DNA损伤后细胞应答反应的重要分子。我们研究小组在前期曾发现NFBD1在DNA损伤后出现转录下调并通过和P53互作而调节细胞凋亡,但NFBD1的转录调控机制仍不清楚,同时试验结果也提示NFBD1很可能参与细胞生长凋亡等的调节。本论文即在此基础上,对NFBD1的转录调控及其调节细胞生长凋亡的功能进行了相对系统的研究。分述如下: (1)NFBD1启动子的克隆、鉴定与初步分析:首先应用5' RACE技术结合生物信息学分析鉴定了NFBD1的转录起始位点,首次发现NFBD1至少存在4种丰度和转录起始位点不同的转录剪接变异体。然后采用PCR定向克隆和酶切亚克隆策略,构建了覆盖NFBD1基因5'侧翼区起始密码子ATG上游5 kb区域的一系列NFBD1启动子荧光素酶报告基因重组体。启动子活性分析表明,NFBD1主要启动子区域定位于其主要转录起始位点区域附近1.5 kb的区域内,同时在其上游还发现了一个活性较低的启动子。采用转录因子结合位点预测分析软件分析表明,NFBD1启动子缺乏TATA盒,但含有典型的CCAAT盒和GC盒以及其它潜在的转录因子结合位点,提示Sp1和NF-Y等转录因子可能参与NFBD1的转录调控。 (2)NFBD1核心启动子区域的鉴定与分析:针对已经初步鉴定的NFBD1主要启动子区域,进一步采用限制性酶切联合DNA blunting方法构建NFBD1启动子荧光素酶报告基因系列删除体。启动子活性分析表明,NFBD1核心启动子区域定位于其主要转录起始位点区域附近325 bp的区域内,该区域分别包含有两个高度保守的Sp1和NF-Y结合位点,但没有发现保守的STAT1结合位点。ChIP和EMSA实验结果表明,Sp1和NF-Y可以与NFBD1启动子在体外(in vitro)和细胞内(in vivo)结合,而STAT1则不与该NFBD1启动子结合。定点突变分析实验结果表明,NFBD1核心启动子区域内NF-Y结合位点的突变对NFBD1启动子活性没有明显影响,但Sp1结合位点的突变导致NFBD1启动子活性的消失,提示Sp1可能在NFBD1的转录调控中起着更为重要的作用。 (3)NFBD1的DNA损伤后转录下调机制研究:采用阿霉素处理A549或HeLa细胞建立DNA损伤细胞模型,发现阿霉素处理后细胞内NFBD1 mRNA和蛋白质水平显著降低,而Sp1的表达水平没有明显变化。磷酸酶处理实验表明,阿霉素处理后Sp1出现磷酸化修饰,同时ChIP实验表明,阿霉素处理导致Sp1与NFBD1启动子的结合活性降低。NFBD1启动子荧光素酶报告基因系列删除体分析实验表明,Sp1参与DNA损伤后的NFBD1转录下调。Sp1的特异性抑制剂光辉霉素A处理和siRNA介导的Sp1表达抑制导致NFBD1启动子活性和内源性NFBD1 mRNA和蛋白质水平的降低,同时,siRNA介导的Sp1表达抑制也提高了细胞对化疗药物阿霉素的敏感性。这些结果表明,Sp1介导DNA损伤后NFBD1的转录下调,这在DNA损伤应答反应中起着重要的作用。 (4)STAT1在NFBD1转录调控中的作用研究:半定量RT-PCR实验结果表明,DNA损伤后NFBD1出现明显的转录下调,STAT1 mRNA水平明显升高,其相应靶基因也同时出现转录激活或抑制。但蛋白质印迹分析实验结果表明,DNA损伤后STAT1蛋白质水平仅呈现轻微升高,且STAT1的磷酸化修饰和转位/移位情况没有明显变化。虽然STAT1抑制剂氟达拉滨处理非特异性地显著降低了NFBD1的启动子活性和mRNA水平,但IFNγ处理诱发的内源性STAT1激活和siRNA介导的STAT1表达抑制对NFBD1的启动子活性和mRNA水平没有明显影响。这些结果表明,STAT1不参与NFBD1的直接转录调控。 (5)NFBD1在细胞生长、凋亡中的作用研究:半定量RT-PCR和蛋白质印迹分析实验结果表明,筛选到的短链NFBD1 siRNA可以成功抑制内源NFBD1的表达,抑制率几乎100%。对HeLa等细胞进行NFBD1 siRNA瞬时转染,发现NFBD1 siRNA可以显著抑制细胞生长。FACS分析和蛋白质印迹分析结果表明,NFBD1 siRNA可以导致细胞内DNA损伤的累积、G2/M期检测点的激活和G2/M期停滞。采用细胞同步化技术,制备不同细胞周期时相的样品,进行NFBD1的基因表达谱分析,发现NFBD1在G2/M期表达水平升高,在G1、S期表达水平降低,表明NFBD1为一G2/M期基因,并参与细胞周期进程或调控。FACS分析结果表明,NFBD1 siRNA瞬时转染导致sub-G1峰的出现,同时蛋白质印迹分析观察到了Caspase 3和PARP的剪接激活,表明NFBD1 siRNA诱发了细胞凋亡。另外蛋白质印迹分析结果还表明,该凋亡过程中P53及其下游靶分子bax和puma也没有明显变化,但noxa在mRNA和蛋白质水平上均显著升高,强烈提示P53非依赖性的Noxa转录激活可能在NFBD1 siRNA诱发细胞凋亡的过程中起着重要作用。另外,MTT分析结果也表明,NFBD1 siRNA显著提高了细胞对阿霉素、顺铂等化疗药物的敏感性。这些结果表明,作为一新的G2/M期基因,NFBD1不仅参与正常的细胞生长,而且参与调节细胞凋亡过程;NFBD1也是一个有效的放化疗增敏剂药物和基因治疗药物靶点。本论文对NFBD1转录调控及其调节细胞生长凋亡的分子功能的系统研究进一步加深了我们对NFBD1分子功能和分子行为机制的认识,将为进一步深入开展NFBD1的基础理论研究、以及促进以NFBD1为分子靶点的抗癌药物开发研究均具有积极的理论和现实意义。
[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 .

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R346

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