本文選題：NDRG2 + Sp1��； 參考：《第四軍醫(yī)大學(xué)》2009年碩士論文

【摘要】： Dynan WS等人于1983最早發(fā)現(xiàn)了轉(zhuǎn)錄因子——Specificity protein 1(Sp1)。Sp1主要特征是含有高度保守的DNA結(jié)合區(qū),即羧基端存在3個(gè)Cys2His2鋅指結(jié)構(gòu)。Sp1是通過其羧基端的鋅指結(jié)構(gòu)與下游靶基因富含GC的序列結(jié)合。 NDRG1(N-Myc down-regulated gene 1)作為NDRG家族最先報(bào)道的成員,在N-Myc敲除的小鼠胚胎中表達(dá)異常增加,所以被命名為N-Myc下游基因1。人NDRG2基因是我室在1999年利用基于PCR消減雜交技術(shù)進(jìn)行神經(jīng)膠質(zhì)瘤與正常腦組織的基因表達(dá)差異研究時(shí)發(fā)現(xiàn)的一個(gè)在多數(shù)腫瘤組織中表達(dá)低的新基因【GenBank登錄號(hào)AF159092】。NDRG2定位于染色體14q11.2,全長(zhǎng)2024bp,含有16個(gè)外顯子,15個(gè)內(nèi)含子,編碼一個(gè)含有357個(gè)氨基酸殘基、分子量約41KDa的蛋白質(zhì)。由于該基因在氨基酸序列上與已發(fā)現(xiàn)的N-Myc下游基因NDRG1(N-myc down-stream regulated gene 1)具有60%同源性,故將其命名NDRG2。 通過生物信息學(xué)分析,我們發(fā)現(xiàn)人NDRG2啟動(dòng)子中含有兩個(gè)髓樣鋅指蛋白1(myeloid zinc finger 1,MZF-1)的潛在結(jié)合位點(diǎn)和四個(gè)Sp1的潛在結(jié)合位點(diǎn)。為了研究Sp1和MZF-1與人NDRG2啟動(dòng)子的關(guān)系,分別將人NDRG2啟動(dòng)子pGL3-NDRG2(-1455——+274)和MZF-1過表達(dá)載體pcDNA3-MZF-1或Sp1過表達(dá)載體pcDNA3- Sp1共轉(zhuǎn)染HEK293細(xì)胞和Hela細(xì)胞。熒光素酶報(bào)告基因分析Sp1促進(jìn)人NDRG2啟動(dòng)子的活性3——4倍,而MZF-1卻無法改變?nèi)薔DRG2啟動(dòng)子活性。接下來,我們進(jìn)一步構(gòu)建Sp1干涉載體pSilencer-Sp1,分別應(yīng)用RT-PCR和Western blot方法檢測(cè)結(jié)果顯示Sp1干涉載體pSilencer-Sp1明顯下調(diào)Sp1的mRNA水平和蛋白質(zhì)水平表達(dá)。我們分別將人NDRG2啟動(dòng)子pGL3-NDRG2(-1455——+274)載體與Sp1顯性負(fù)突變載體PEBGN-Sp1或Sp1的干涉載體pSilencer-Sp1共轉(zhuǎn)染HEK293細(xì)胞和Hela細(xì)胞。熒光素酶報(bào)告基因分析,Sp1顯性負(fù)突變體PEBGN-Sp1和Sp1干涉載體pSilencer-Sp1明顯抑制人NDRG2啟動(dòng)子的活性。 為了明確Sp1是否與人NDRG2啟動(dòng)子pGL3-NDRG2(-1455——+274)結(jié)合,我們將一系列不同人NDRG2啟動(dòng)子的截短體與陰性對(duì)照載體pcDNA3或Sp1過表達(dá)載體pcDNA3- Sp1共轉(zhuǎn)染HEK293細(xì)胞。結(jié)果顯示Sp1可能與人NDRG2啟動(dòng)子中三個(gè)富含GC的區(qū)域結(jié)合,分別為人NDRG2啟動(dòng)子A:NDRG2(-148——-138)、B:NDRG2(+15——+19)、C:NDRG2(+29——+33)。接下來,我們通過PCR的方法構(gòu)建了一系列Sp1結(jié)合位點(diǎn)突變的人NDRG2啟動(dòng)子的載體——M1(A點(diǎn)突變)、M2(B點(diǎn)突變)、M3(C點(diǎn)突變)、M12(AB點(diǎn)突變)、M13(AC點(diǎn)突變)和M23(BC點(diǎn)突變)。熒光素酶報(bào)告基因分析,單個(gè)Sp1結(jié)合位點(diǎn)突變體M1、M2和M3相對(duì)于野生型的啟動(dòng)子活性均有不同程度的降低。兩個(gè)Sp1結(jié)合位點(diǎn)突變載體M12、M13和M23相對(duì)于野生型的啟動(dòng)子活性至少降低約50%。但是以上結(jié)果都無法證明Sp1是如何調(diào)控人NDRG2啟動(dòng)子,為了進(jìn)一步研究Sp1是否與人NDRG2啟動(dòng)子直接結(jié)合?我們通過體內(nèi)實(shí)驗(yàn)染色體免疫共沉淀(ChIP)和體外實(shí)驗(yàn)?zāi)z遷移實(shí)驗(yàn)(EMSA),進(jìn)一步實(shí)驗(yàn)證實(shí)了Sp1與人NDRG2啟動(dòng)子(-148——-138)直接結(jié)合。 綜上所述,我們發(fā)現(xiàn)NDRG2是Sp1轉(zhuǎn)錄調(diào)控的一個(gè)新的靶基因。Sp1對(duì)NDRG2轉(zhuǎn)錄調(diào)控的新認(rèn)識(shí),不僅豐富了Sp1的信號(hào)轉(zhuǎn)導(dǎo)通路,而且為進(jìn)一步明確NDRG2的功能、為NDRG2在腫瘤治療中的應(yīng)用提供了新的線索和實(shí)驗(yàn)依據(jù)。
[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 .

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 鄧艷春,藥立波,劉新平,聶曉燕,王吉村,張曉光,蘇成芝;人腦內(nèi)一含有ACP樣結(jié)構(gòu)域新基因的發(fā)現(xiàn)[J];生物化學(xué)與生物物理進(jìn)展;2001年01期

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本文編號(hào)：1761215