【作者】 陈蔚文；

【导师】 张建业；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 近年来随着人口老龄化及饮食结构的改变,前列腺癌的发病率和死亡率有迅速上升的趋势,探索前列腺癌的综合治疗方法尤其是基因治疗已日益受到重视。AMACR是新近发现的前列腺癌高度相关的基因,本课题对其在前列腺癌细胞中的表达调控机制进行了研究。AMACR是通过cDNA消减文库和高通量微列阵筛选技术,从良、恶性前列腺组织中鉴别的人类前列腺癌及组织特异性基因之一,它在＞70%的前列腺上皮内瘤中度表达、＞95%的初期前列腺癌中高度表达,在正常前列腺中仅有少量表达或无法检出。其表达产物主要参与支链脂肪酸及其衍生物的β氧化代谢和胆汁酸的生物合成,是检测前列腺癌的新型阳性分子标记物,可作为现有前列腺癌诊断方法的重要补充。除了作为一个重要的诊断指标,AMACR与前列腺癌的发生和罹患风险、分化程度均存在一定关系:在体外刺激AMACR过表达有利于前列腺癌细胞的最佳生长;利用RNAi技术沉默AMACR基因则可抑制前列腺癌细胞株的生长,均暗示了AMACR在前列腺癌形成和发展过程中的潜在作用,有可能为前列腺癌的基因治疗提供新的线索。因此阐明AMACR在前列腺癌中过表达的机制对于进一步了解前列腺癌的发生发展机制,探索新的治疗途径有重要意义。来自cDNA芯片和实时PCR的研究结果说明mRNA水平的上调是AMACR在前列腺癌过表达的重要原因。本研究克隆了AMACR 2315 bp（-2252～+63）的启动子序列,插入荧光素酶报道基因表达载体pGL3-Basic,在AMACR表达水平最强的LNCaP细胞中检测了2315bp AMACR启动子的活性。继而利用瞬时转染分析,报道基因分析和电泳迁移率改变分析（EMSA）、综合性突变分析等技术在2315 bp的AMACR启动子中鉴定了一个20-bp功能性正调控序列;利用共转染分析、突变分析、报道基因分析、RT-PCR、Western blot以及EMSA等研究方法发现抑癌基因p53可能通过间接抑制AMACR启动子中的CPBP元件来抑制AMACR的表达。第一部分人AMACR基因启动子的克隆、鉴定及初步分析【研究目的】克隆人AMACR基因5’上游2315 bp启动调控区片段,测定其启动子活性,并对其进行初步分析。【研究方法】①提取人基因组DNA,PCR扩增AMACR基因5’上游2315 bp（-2252～+63）启动调控区序列,插入pGL3-Basic,构建AMACR启动子-荧光素酶报道基因表达载体pGL3-2315,酶切鉴定和测序分析证实插入是否正确。②pGL3-2315瞬时转染前列腺癌细胞LNCaP和PC-3,荧光素酶报道基因分析检测启动子活性。③利用Erase-a-Base System,在pGL3-2315基础上构建5’-缺失突变体库,选取11个较大范围的缺失突变体,瞬时转染LNCaP细胞,报道基因分析观察较大范围的缺失突变对启动子活性的影响。④将各种蛋白因子表达载体与pGL3-2315共转染LNCaP和PC-3细胞,报道基因分析观察各种蛋白因子对启动子活性的影响。⑤pGL3-2315瞬时转染LNCaP细胞,或与pSG5-hAR共转染PC-3细胞后,加入不同浓度的雄激素类似物R1881处理24h,报道基因分析观察R1881对启动子活性的影响。【结果】①重组质粒pGL3-2315酶切鉴定及DNA测序结果均正确。②pGL3-2315在LNCaP细胞呈现很强的启动子活性,在PC-3细胞呈现明显的启动子活性。③pGL3-2315的缺失突变分析显示-423～-93 330 bp区域的缺失可导致启动子活性下降70%。④共转染实验结果显示PPARγ₂,PTEN,Sp1,ras和myc的表达载体对2315 bp片段的活性没有明显影响。而p53、C/EBPα、NF-κB p50的表达载体明显抑制AMACR启动子活性。⑤雄激素类似物R1881对pGL3-2315的启动子活性无明显影响。【结论】正确克隆了人前列腺癌相关基因AMACR上游2315 bp（-2252～+63）的启动子片段,该2315 bp片段在LNCaP细胞中呈现了很强的启动子活性。2315 bp片段中存在一个明显的功能性正调控区域（-423～-93）。转录因子p53,C/EBPα和NF-κB p50过表达明显抑制AMACR启动子活性。AMACR启动子活性不受雄激素信号途径调控。第二部分AMACR基因启动子中一个20-bp正调控序列的鉴定【研究目的】在AMACR启动子-423～-93区鉴定具有正调控作用的DNA元件/序列,为进一步研究AMACR基因在前列癌细胞中过表达的转录调控机制奠定基础。【研究方法】①利用Erase-a-Base System建立的缺失突变体库,在pGL3-486（-423）区域内筛选更为精细的缺失突变体,瞬时转染LNCaP细胞,报道基因分析观察启动子活性的变化。②人工合成缺失突变分析发现的20-bp正调控序列,插入异源启动子-荧光素酶报道基因载体pGL3-Promoter中SV40启动子的上游和pGL3-maspin中maspin启动子的上游,瞬时转染LNCaP细胞,报道基因分析观测20-bp正调控序列对异源启动子的作用。③采用PCR连接反应在pGL3-486中删除该20-bp序列,构建内缺失突变体pGL3-486id,瞬时转染LNCaP细胞,报道基因分析观察启动子活性的变化。④采用EMSA检测20-bp正调控序列的特异性结合蛋白。人工合成20-bp正调控序列,以末端转移酶将其末端标记地高辛;提取LNCaP细胞核蛋白,与地高辛标记的20-bp探针进行结合反应,同时进行各种特异性和非特异性竞争结合反应,进行聚丙烯酰胺凝胶电泳,检测电泳迁移率的改变。⑤人工合成20-bp正调控序列作为陷阱DNA。以50～300倍过量的陷阱DNA与pGL3-2315共转染LNCaP细胞,测定双荧光素酶活性,观察20-bp正调控序列陷阱DNA对AMACR启动子活性的影响。【结果】①-136～-117片段的缺失使启动子活性下降了3.8倍,表明该20 bp片段为一正调控序列。②pGL3-486中该20-bp正调控序列的内缺失导致启动子活性下降了67%。③该20-bp正调控序列插入异源启动子SV40和mspin上游,导致SV40和mspin启动子活性分别升高了4.3倍和3.3倍。④利用LNCaP核蛋白提取液检测到了该20-bp序列的电泳阻滞带,可被自身竞争性抑制,但不能被异源序列ARE元件和突变的20-bp序列竞争性抑制。⑤20-bp正调控序列陷阱DNA可明显抑制pGL3-2315的启动子活性,该抑制作用随剂量升高而加强,至200倍过量时达到高峰,可使2315 bp启动子活性降低63%。【结论】在AMACR基因上游启动子-136～-117区鉴定到一个20-bp功能性正调控序列,对AMACR基因在前列腺癌细胞LNCaP中的转录激活有重要作用。在LNCaP核蛋白提取液中检测到与此正调控序列特异结合的蛋白质。进一步的研究将对此结合蛋白进行分离、鉴定,研究其调控机制。第三部分p53对LNCaP细胞中AMACR表达的抑制作用【研究目的】研究p53对LNCaP细胞中AMACR表达的抑制作用,并探讨其作用机制。【研究方法】①以不同剂量的pCMV-p53或pCMV-p53DC瞬时转染LNCaP细胞,利用报道基因分析检测p53过表达对AMACR启动子活性的影响。②以不同剂量的pCMV-p53或pCMV-p53DC瞬时转染LNCaP细胞48h,收集细胞,以RT-PCR、Western blot观察p53过表达在mRNA和蛋白水平对AMACR表达的影响。③pCMV-p53与pGL3-2315及缺失突变体pGL3-486、pGL3-140分别共转染LNCaP细胞,报道基因分析观测启动子活性的变化,大致确定p53在启动子上的间接作用区域。④分析-77至+63（pGL3-140）区内两个潜在的正调控CPBP元件（CPBP1/2）的功能。人工合成CPBP1/2序列,插入异源启动子SV40和maspin上游,瞬时转染LNCaP细胞,报道基因分析观察启动子活性的变化;将50～300倍的CPBP1/2序列作为陷阱DNA与pGL3-2315共转染LNCaP细胞,报道基因分析观察CPBP1/2序列竞争对2315 bp启动子活性的影响。地高辛标记CPBP1序列末端作为探针,提取LNCaP细胞核蛋白,EMSA检测CPBP1序列特异性结合蛋白。⑤确定p53过表达对CPBP1序列的间接性抑制作用。将pCMV、pCMV-p53或pCMV-p53DC分别与CPBP1-SV40启动子-荧光素酶报道基因载体共转染LNCaP细胞,报道基因分析观察SV40启动子活性的变化。分别提取经pCMV、pCMV-p53或pCMV-p53DC转染的LNCaP核蛋白,与地高辛标记的CPBP1探针进行EMSA,观察外源性p53过表达对CPBP1元件结合活性的抑制作用。【结果】①pCMV-p53而非pCMV-p53DC转染可抑制LNCaP细胞中AMACR启动子的活性,该抑制作用呈剂量依赖性。②pCMV-p53而非pCMV-p53DC转染可在mRNA和蛋白水平抑制LNCaP细胞中AMACR的表达,该抑制作用呈剂量依赖性。③p53对AMACR启动子的抑制作用区域在-77～+63（pGL3-140）,MatInspector 2.2在此区域内检索到两个潜在的正调控CPBP元件（CPBP1/2）。④CPBP1序列可使SV40启动子活性升高4.2倍,maspin启动子活性升高2.4倍,而CPBP2序列仅有微弱的增强作用。过量的CPBP1序列陷阱DNA可导致pGL3-2315活性明显下降,而CPBP2元件没有明显作用。CPBP1元件可与LNCaP核蛋白形成明显的阻滞带,该阻滞带可被自身竞争性抑制以及特异性抗体anti-CPBP大部阻断。⑤pCMV-p53而非pCMV-p53DC转染可抑制CPBP1-SV40异源启动子活性。经pCMV-53而非pCMV-p53DC转染的LNCaP核蛋白与CPBP1元件形成的的电泳阻滞带明显减弱。【结论】p53可能通过抑制AMACR启动子中正调控CPBP元件的结合活性,抑制LNCaP细胞中AMACR的表达。更多还原

【Abstract】 Recent years,the incidence and fatality of prostate cancer is upgraded quickly with the aging of population structure and the composition change of food.It is increasingly important to explore the combined therapy especially gene therapy for prostate cancer.AMACR is a prostate cancer-related gene found recently,whose transcriptional regulation in prostate cancer cells is studied in this work.AMACR is an enzyme involved inβ-oxidation of branched-chain fatty acids and bile acid intermediates.Normal prostate epithelial and stromal cells typically show little expression of AMACR.In contrast,＞95%of the primary prostate cancer cases show strong expression of AMACR protein,with＞70%of the precancerous lesions, high-grade prostatic intraepithelial hyperplasia,expressing AMACR at intermediate level.The remarkable consistence and specificity of AMACR overexpression in prostate cancer has made it a promising new diagnostic marker for this disease. Recent studies also imply that elevated AMACR expression is functionally important for optimal growth of prostate cancer cells in vitro,and Small interference RNA （siRNA）against AMACR reduced the expression of AMACR and significantly impaired proliferation of the androgen-responsive prostate cancer cell line LAPC-4, suggesting novel therapeutic strategies based on this gene and/or pathway.Despite this interest,little is known about the transcriptional regulation of AMACR in normal tissue as well as in prostate cancer.Although it is possible that protein stability might contribute to the enhanced AMACR protein level seen in clinical samples,the fact that overexpression of AMACR was first detected by cDNA microarray studies and quantitative PCR analyses revealed large increases at the mRNA level clearly indicated that a significant part of AMACR overexpression could be attributed to increased mRNA level.To begin to understand the transcriptional regulation of the AMACR gene,we cloned the luciferase reporter gene expression vector including 2315 bp AMACR promoter,then we use nucleic acid mutation technique,reporter gene assay,RT-PCR, Western blot and electrophoretic mobility shift assay（EMSA）to define a 20-bp positive-regualtory sequence in the AMACR promoter.Finally,we found tumor suppressor gene p53 inhibited the expression of AMACR in LNCaP cells by repressing the binding activity of a positive-regulatory CPBP element in promoter of AMACR indirectly.PART ONE:CLONING,IDENTIFICATION AND PRELIMINARY ANALYSIS OF HUMAN AMACR PROMOTERObjective:The aim of this part is to clone 2315 bp promoter fragment upstream of AMACR gene,to determine its promoter activity and to make a preliminary analysis on it.Methods:①According to the sequence of AMACR gene in Genbank,a pair of primer was designed for PCR.2315 bp promoter fragment of AMACR gene was amplified by PCR and was inserted into pGL3-Basic,a promoter-less luciferase reporter vector,to form 2315 bp AMACR promoter-luciferase reporter plasmid（pGL3-2315）that proved to be right by the restriction enzyme digestion and DNA sequencing. ②pGL3-2315 was cotransfected into prostate cancer cell lines LNCaP and PC-3 with pRL-TK using FuGENE HD,and then the expression of luciferase reporter driven by 2315 bp promoter of AMACR was tested by luciferase activity assay.③A series of 5’ deletion mutants-luciferase reporter plasmids of 2315 bp promoter were obtained using Erase-a-Base System based on pGL3-2315,and then 11 mutants of which were selected to be transfected into LNCaP cells respectively in order to observe the effect of deletion mutation on promoter activity of AMACR.④The pGL3-2315 was cotransfected into LNCaP and PC-3 cells with the expression vectors of C/EBPα,PPARγ₂,PTEN,NF-κB p50,Sp1,p53, ras or myc,and then the change of promoter activity was observed by luciferase activity assay.⑤To observe whether or not 2315 bp promoter is regulated by androgen, the pGL3-2315 was transfected into LNCaP cells,or was co-transfected with pSG5-hAR into PC-3 cells.Then both LNCaP and PC-3 cells were treated with 10^-10～10^-6M R1881 in 2%charcoal treated FBS-RPMI 1640 medium for 24 h.The promoter activities were observed by luciferase activity assay.Results:①The recombinant plasmid pGL3-2315 was proved to be right by the restriction enzyme digestion and DNA sequencing.②The 2315 bp promoter fragment presented a strong promoter activity in LNCaP cell line and a significant promoter activity in PC-3 cell line respectively.③The deletion from -423 to -93 reduced the promoter activity 70%.④Co-transfection with expression vector of PPARγ₂,PTEN,Sp1,ras or myc has no significant effect on AMACR promoter activity.However, co-transfection with expression vectors of p53,C/EBPαor NF-κB p50 significantly inhibited the AMACR promoter activity.⑤R1881 had no significant effect on promoter activity of pGL3-2315 in both LNCaP cell line and PC-3 cell lineConclusion:The 2315 bp（-2252～+63）promoter fragment upstream of the human prostate cancer-related gene AMACR was cloned successfully.This 2315 bp fragment presented strong promoter activity in LNCaP cells.A significant functional positive-regulatory region（-423～-93）was found within 2315 bp promoter fragment of AMACR.Overexpression of transcriptional factors,p53,C/EBPαor NF-κB p50 significantly inhibited the promoter activity of AMACR.The AMACR promoter activity was independent of the regulation of androgen signal pathway.PART TWO:IDENTIFICATION OF A 20-BP POSITIVE-REGULATORY SEQUENCE IN AMACR PROMOTERObjective:To identify the positive-regualtory element/sequence within the positive-regulatory region（-423bp～-93bp）in AMACR promoter.It will provide an insight into the regulatory mechanism of AMACR gene overexpression in prostate cancer cells in further study.Methods:①A series of refined 5’ deletion mutants within -423 promoter fragment （pGL3-486）were further obtained from the 5’ deletion mutants library established by Erase-a-Base System based on pGL3-2315.All of them were sequenced and then were transiently transfected into LNCaP cells respectively to observe the promoter activity change by lucuferase activity assay.②The 20-bp positive-regulatory sequence was synthesized and inserted into the upstream of SV40 promoter in pGL3-promoter and maspin promoter in pGL3-maspin respectively.Then the recombinant 20-bp-heterologous promoter-luciferase reporter gene plasmids were transiently transfected into LNCaP cells,and the effects of 20-bp positive-regulatory sequence on heterologous promoters were test by reporter gene assay.②The 20-bp positive-regulatory sequence was deleted from pGL3-486 using PCR ligation method to construct internal deletion mutant, pGL3-486id.The pGL3-486id was transiently transfected into LNCaP cells,and then the change of promoter activity of this internal deletion mutant was detected by reporter gene assay.④Electrophoresis mobility shift assay（EMSA）was used to identify the specific binding protein to the 20-bp positive-regulatory sequence. 20-bp positive-regulatory sequence was synthesized and labeled with digoxigenin by terminal transferase.The nuclear protein was extracted from LNCaP cells and bound to the labeled probe of 20-bp sequence with unlabeled specific-competitive or unspecific-competitive oligonucleotides.The binding complexes run in polyacryl-amide gel to observe electrophoresis mobility shift.⑤The 20-bp sequence was synthesized as decoy DNA.The pGL3-2315 was co-transfected with 50-fold to 300-fold excess decoy DNA of 20-bp positive-regulatory sequence into LNCaP cells,and then the effect of decoy DNA on promoter activity of pGL3-2315 was observed by luciferase activity assay. Results:①Deletion from-136 to-117 decreased the promoter activity 3.8-fold, suggesting that this 20 bp fragment is a positive-regulatory sequence.②The internal deletion of 20-bp positive-regulatory sequence from pGL3-486 result in the promoter activity decreaseing 67%.③This 20-bp positive-regulatory sequence was inserted into the upstream of heterologous promoters,SV40 and mspin,result in heterologous the promoter activities increasing 4.3-fold and 3.3-fold respectively.④A sequence-specific binding protein to 20-bp positive-regulatory sequence was identified from LNCaP nuclear extracts by EMSA.This shift band can be blocked by a 125-fold excess amount of unlabeled 20-bp probe,but not the heterologous probe ARE or mutated 20-bp probe.⑤The promoter activity of pGL3-2315 was inhibited significantly by co-transfecting with different excess amounts of 20-bp positive-regulatory sequence decoy DNA.This inhibition effect was dose-dependent,300-fold excess amount of the 20-bp positive-regulatory sequence decoy DNA decreased the promoter activity about 63%.Conclusion:A 20-bp functional positive-regulatory sequence,which played a critical role in transactivation of AMACR gene in prostate cancer cell LNCaP, was identified from-136 to-117 in promoter of AMACR,and a sequence-specific binding protein to the 20-bp positive-regulatory sequence was detected from nuclear extract of LNCaP cells using EMSA. Isolation,identification and regulatory mechanism research of the sequence-specific binding protein to the 20-bp positive-regulatory sequence will be done in further work. PART THREE:p53 INHIBITED THE EXPRESSION OF AMACR IN LNCaP CELLSObjective:It is aimed to confirm the repressing effect of p53 overexpression on AMACR expression in LNCaP cells and to research the mechanism of p53 inhibiting AMACR expression.Methods:①A series doses of pCMV-p53 or pCMV-p53DC were transfected into LNCaP cells,then the effects of p53 overexpression on AMACR promoter activity were observed by reporter gene assay.②A series doses of pCMV-p53 or pCMV-p53DC were transfected into LNCaP and PC-3 cells,then the effects of p53 overexpression on AMACR expression at mRNA and protein level were observed by RT-PCR and Western blot respectively.③pGL3-2315 as well as its deletion mutants,pGL3-486 and pGL3-140, was co-transfected with pCMV-p53 into LNCaP cells respectively,then the change of promoter activity was observed by reporter gene assay to determine the region inhibited by p53 indirectly in AMACR promoter.④To analyze the function of two potential positive CPBP element （CPBP1/2）within-77 to +63（pGL3-140）,the CPBP1/2 sequence was synthesized and inserted into the upstream of heterologous promoters, SV40 and mspin.Then the recombinant plasmids were transiently transfected into LNCaP cells,and the change of promoter activity was observed by reporter gene assay.The pGL3-2315 was co-transfected with 50-fold to 300-fold excess amount of CPBP1/2 sequence decoy DNA into LNCaP cells,and the effects of CPBP1/2 sequence competition on 2315 bp promoter activity was observed by reporter gene assay.CPBP1 sequence was labeled with digoxigenin with terminal transferase.The nucleic extracts were extracted from LNCaP cells and EMSA was used to identify the sequence-specific binding protein to the CPBP1 sequence.⑤To confirm the indirect repression on CPBP1 sequence by p53 overexpression,the recombinant luciferase repoter gene plasmid containing CPBP1-SV40 promoter was co-transfected with pCMV, pCMV-p53 or pCMV-p53DC respectively into LNCaP cells,then the change of promoter activity was observed by reporter gene assay. Moreover,the nuclear protein was extracted from LNCaP cells that transfected with pCMV,pCMV-p53 or pCMV-p53DC respectively, then the EMSA was performed with CPBP1 probe labeled by digoxigenin to observe the repressing effect on CPBP1 sequnce by p53 overexpression.Results:①p53 but not the p53DC overexpression inhibited the activity of AMACR promoter in a dose-dependent way in LNCaP cells.②p53 but not the p53DC overexpression inhibited expression of AMACR at transcriptional level and protein level in a dose-dependent way in LNCaP cells and PC-3 cells.③The region in AMACR promoter inhibited indirectly by p53 overexpression located from-77 to +63,within which there are two potential positive-regulatory CPBP elements by MatInspector 2.2 search.④The heterologous promoter activity increased 4.2-fold（SV40 promter）or 2.4-fold（maspin promoter）by insertion of CPBP1 sequence into its upstream,but CPBP2 sequence only presented minimal enhancement. Excess amount of CPBP1 sequnce decoy DNA but not the CPBP2 decreased the promoter activity of pGL3-2315 significantly.The digoxigenin labeled CPBP1 probe formed significant DNA-protein complex with nuclear extract from LNCaP cells.This shif band could be competed by 125-fold excess amount of unlabeled CPBP1 probe,or be blocked largely by specific anti-CPBP.⑤The transfection of pCMV-p53 but not the pCMV-p53DC inhibited the activity of CPBP1-SV40 heterologous promoter.The nuclear extract from LNCaP cells transfected with pCMV-p53 but not the pCMV-p53DC decreased the shift band formed with CPBP1 probe.Conclusion:The p53 probably inhibit the AMACR expression by inhibiting the binding activity of CPBP element in promoter of AMACR gene.更多还原