转录因子Sp1调控人NDRG2转录机制研究

发布时间：2018-04-17 00:17

  本文选题：NDRG2 + Sp1　； 参考：《第四军医大学》2009年硕士论文

【摘要】： Dynan WS等人于1983最早发现了转录因子——Specificity protein 1(Sp1)。Sp1主要特征是含有高度保守的DNA结合区,即羧基端存在3个Cys2His2锌指结构。Sp1是通过其羧基端的锌指结构与下游靶基因富含GC的序列结合。 NDRG1(N-Myc down-regulated gene 1)作为NDRG家族最先报道的成员,在N-Myc敲除的小鼠胚胎中表达异常增加,所以被命名为N-Myc下游基因1。人NDRG2基因是我室在1999年利用基于PCR消减杂交技术进行神经胶质瘤与正常脑组织的基因表达差异研究时发现的一个在多数肿瘤组织中表达低的新基因【GenBank登录号AF159092】。NDRG2定位于染色体14q11.2,全长2024bp,含有16个外显子,15个内含子,编码一个含有357个氨基酸残基、分子量约41KDa的蛋白质。由于该基因在氨基酸序列上与已发现的N-Myc下游基因NDRG1(N-myc down-stream regulated gene 1)具有60%同源性,故将其命名NDRG2。 通过生物信息学分析,我们发现人NDRG2启动子中含有两个髓样锌指蛋白1(myeloid zinc finger 1,MZF-1)的潜在结合位点和四个Sp1的潜在结合位点。为了研究Sp1和MZF-1与人NDRG2启动子的关系,分别将人NDRG2启动子pGL3-NDRG2(-1455——+274)和MZF-1过表达载体pcDNA3-MZF-1或Sp1过表达载体pcDNA3- Sp1共转染HEK293细胞和Hela细胞。荧光素酶报告基因分析Sp1促进人NDRG2启动子的活性3——4倍,而MZF-1却无法改变人NDRG2启动子活性。接下来,我们进一步构建Sp1干涉载体pSilencer-Sp1,分别应用RT-PCR和Western blot方法检测结果显示Sp1干涉载体pSilencer-Sp1明显下调Sp1的mRNA水平和蛋白质水平表达。我们分别将人NDRG2启动子pGL3-NDRG2(-1455——+274)载体与Sp1显性负突变载体PEBGN-Sp1或Sp1的干涉载体pSilencer-Sp1共转染HEK293细胞和Hela细胞。荧光素酶报告基因分析,Sp1显性负突变体PEBGN-Sp1和Sp1干涉载体pSilencer-Sp1明显抑制人NDRG2启动子的活性。 为了明确Sp1是否与人NDRG2启动子pGL3-NDRG2(-1455——+274)结合,我们将一系列不同人NDRG2启动子的截短体与阴性对照载体pcDNA3或Sp1过表达载体pcDNA3- Sp1共转染HEK293细胞。结果显示Sp1可能与人NDRG2启动子中三个富含GC的区域结合,分别为人NDRG2启动子A:NDRG2(-148——-138)、B:NDRG2(+15——+19)、C:NDRG2(+29——+33)。接下来,我们通过PCR的方法构建了一系列Sp1结合位点突变的人NDRG2启动子的载体——M1(A点突变)、M2(B点突变)、M3(C点突变)、M12(AB点突变)、M13(AC点突变)和M23(BC点突变)。荧光素酶报告基因分析,单个Sp1结合位点突变体M1、M2和M3相对于野生型的启动子活性均有不同程度的降低。两个Sp1结合位点突变载体M12、M13和M23相对于野生型的启动子活性至少降低约50%。但是以上结果都无法证明Sp1是如何调控人NDRG2启动子,为了进一步研究Sp1是否与人NDRG2启动子直接结合?我们通过体内实验染色体免疫共沉淀(ChIP)和体外实验凝胶迁移实验(EMSA),进一步实验证实了Sp1与人NDRG2启动子(-148——-138)直接结合。 综上所述,我们发现NDRG2是Sp1转录调控的一个新的靶基因。Sp1对NDRG2转录调控的新认识,不仅丰富了Sp1的信号转导通路,而且为进一步明确NDRG2的功能、为NDRG2在肿瘤治疗中的应用提供了新的线索和实验依据。
[Abstract]:Sp1 is a highly conserved DNA binding region , that is , there are three Cys2His2 zinc finger structures in the carboxy terminal . Sp1 is a GC - rich sequence linked to the downstream target gene through the zinc finger structure at its carboxy terminus .



NDRG1 ( N - Myc down - regulated gene 1 ) , as the first member of NDRG family , was named N - Myc downstream gene 1.Human NDRG2 gene was named N - Myc downstream gene 1.Human NDRG2 gene was a new gene expressed in most tumor tissues by using PCR - based subtractive hybridization technique . NDRG2 was located at chromosome 14q11.2 with a full length of 2024bp , containing 16 exons and 15 intron , encoding a protein containing 357 amino acid residues and a molecular weight of about 41 kDa . Since the gene had 60 % homology with the N - myc down - stream regulated gene 1 found on the amino acid sequence , NDRG2 was named NDRG2 .



In order to study the relationship between Sp1 and MZF - 1 and human NDRG2 promoter , the expression vector pcDNA3 - MZF - 1 or Sp1 overexpression vector pcDNA3 - MZF - 1 or Sp1 overexpression vector pcDNA3 - Sp1 was co - transfected with the expression vector pcDNA3 - MZF - 1 or Sp1 overexpression vector pcDNA3 - Sp1 .



In order to clarify whether Sp1 was co - transfected with human NDRG2 promoter pGL3 - NDRG2 ( -764274 ) , a series of human NDRG2 promoters were co - transfected with pcDNA3 or Sp1 overexpression vector pcDNA3 - Sp1 . The results showed that Sp1 could bind to three GC - rich regions of human NDRG2 promoter . The results showed that Sp1 could be directly combined with human NDRG2 promoter . In order to further study whether Sp1 was directly bound to human NDRG2 promoter , we confirmed that Sp1 was directly linked to human NDRG2 promoter ( -148 _ -138 ) .



In conclusion , we find that NDRG2 is a new target gene for transcription regulation of Sp1 . Sp1 has a new understanding of transcription regulation of NDRG2 , which not only enriches the signal transduction pathway of Sp1 , but also provides a new clue and experimental basis for the application of NDRG2 in tumor therapy .

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

【参考文献】

相关期刊论文 前1条

1 邓艳春,药立波,刘新平,聂晓燕,王吉村,张晓光,苏成芝;人脑内一含有ACP样结构域新基因的发现[J];生物化学与生物物理进展;2001年01期

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