| 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.
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