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家族最先報道的成員,在N-Myc敲除的小鼠胚胎中表達異常增加,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)進行神經(jīng)膠質(zhì)瘤與正常腦組織的基因表達差異研究時發(fā)現(xiàn)的一個在多數(shù)腫瘤組織中表達低的新基因【GenBank登錄號AF159092】。NDRG2定位于染色體14q11.2,全長2024bp,含有16個外顯子,15個內(nèi)含子,編碼一個含有357個氨基酸殘基、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啟動子中含有兩個髓樣鋅指蛋白1(myeloid zinc finger 1,MZF-1)的潛在結(jié)合位點和四個Sp1的潛在結(jié)合位點。為了研究Sp1和MZF-1與人NDRG2啟動子的關(guān)系,分別將人NDRG2啟動子pGL3-NDRG2(-1455——+274)和MZF-1過表達載體pcDNA3-MZF-1或Sp1過表達載體pcDNA3- Sp1共轉(zhuǎn)染HEK293細胞和Hela細胞。熒光素酶報告基因分析Sp1促進人NDRG2啟動子的活性3——4倍,而MZF-1卻無法改變?nèi)薔DRG2啟動子活性。接下來,我們進一步構(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。我們分別將人NDRG2啟動子pGL3-NDRG2(-1455——+274)載體與Sp1顯性負突變載體PEBGN-Sp1或Sp1的干涉載體pSilencer-Sp1共轉(zhuǎn)染HEK293細胞和Hela細胞。熒光素酶報告基因分析,Sp1顯性負突變體PEBGN-Sp1和Sp1干涉載體pSilencer-Sp1明顯抑制人NDRG2啟動子的活性。 為了明確Sp1是否與人NDRG2啟動子pGL3-NDRG2(-1455——+274)結(jié)合,我們將一系列不同人NDRG2啟動子的截短體與陰性對照載體pcDNA3或Sp1過表達載體pcDNA3- Sp1共轉(zhuǎn)染HEK293細胞。結(jié)果顯示Sp1可能與人NDRG2啟動子中三個富含GC的區(qū)域結(jié)合,分別為人NDRG2啟動子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)合位點突變的人NDRG2啟動子的載體——M1(A點突變)、M2(B點突變)、M3(C點突變)、M12(AB點突變)、M13(AC點突變)和M23(BC點突變)。熒光素酶報告基因分析,單個Sp1結(jié)合位點突變體M1、M2和M3相對于野生型的啟動子活性均有不同程度的降低。兩個Sp1結(jié)合位點突變載體M12、M13和M23相對于野生型的啟動子活性至少降低約50%。但是以上結(jié)果都無法證明Sp1是如何調(diào)控人NDRG2啟動子,為了進一步研究Sp1是否與人NDRG2啟動子直接結(jié)合?我們通過體內(nèi)實驗染色體免疫共沉淀(ChIP)和體外實驗?zāi)z遷移實驗(EMSA),進一步實驗證實了Sp1與人NDRG2啟動子(-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)控的一個新的靶基因。Sp1對NDRG2轉(zhuǎn)錄調(diào)控的新認識,不僅豐富了Sp1的信號轉(zhuǎn)導(dǎo)通路,而且為進一步明確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驗依據(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)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R346

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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)與生物物理進展;2001年01期

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本文編號：1761215