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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳小梨(Show-Li Chen) | |
dc.contributor.author | Pei-Hong Chen | en |
dc.contributor.author | 陳沛鴻 | zh_TW |
dc.date.accessioned | 2021-06-13T15:43:30Z | - |
dc.date.available | 2013-08-13 | |
dc.date.copyright | 2008-08-13 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-07-03 | |
dc.identifier.citation | 1. Solit, D.B., Scher, H.I. and Rosen, N. (2003) Hsp90 as a therapeutic target in prostate cancer. Semin Oncol, 30, 709-716.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37781 | - |
dc.description.abstract | 攝護腺癌是造成全球男性癌症死亡因素的第二位。雄激素和其受體間的訊息傳導被認為是造成攝護腺癌進程的主要起因,雄激素受體的輔助活化因子在雄激素訊息傳導當中扮演了舉足輕重的角色並且在一些攝護腺癌組織中被大量地表現出來。雄激素與其受體結合後作用在啟動子的雄激素反應元素上而活化目標基因(例如p21、FGF8等),進而刺激細胞複製並促進最後腫瘤的形成。
我們實驗室最近利用酵母菌雙雜合系統的分析,以雄激素受體的羧基端當作誘餌,發現了核感受器交互作用蛋白質。核感受器交互作用蛋白質為一新的雄激素受體輔助活化因子,可以藉著雄激素的作用去增強活化血清攝護腺特異抗原的表現。在依賴雄激素受體存活的攝護腺癌細胞株LNCaP的細胞裡面,核感受器交互作用蛋白質能與雄激素受體直接地結合,其功能參與了細胞的複製與生存。利用小干擾核糖核酸減少核感受器交互作用蛋白質的表現,能降低雄激素受體目標基因的活化,結果導致細胞的凋亡。LNCaP細胞對雄激素受體調控的敏感,使得核感受器交互作用蛋白質的表現量隨著雄激素的濃度而增加。 在這份論文中,我們進一步調查核感受器交互作用蛋白質基因的調控機轉和其在雄激素受體蛋白質穩定上所扮演的角色。我們發現核感受器交互作用蛋白質是一個新的雄激素受體目標基因,並且其功能對雄激素受體的訊息傳導和攝護腺癌細胞株的調控皆佔有一席之地。核感受器交互作用蛋白質的核心啟動子區域範圍坐落在轉錄起始點上游-413到+94之間,其去氧核糖核酸序列由荷爾蒙反應元素和三個似Sp1結合區所組成。核感受器交互作用蛋白質的啟動子富有G和C元素,沒有典型的TATA盒子區,由Sp1轉錄因子所調控。核感受器交互作用蛋白質在LNCaP細胞裡隨著雄激素濃度的提升而增加;並且在缺乏雄激素受體表現的293T細胞中,當以外送質體表現雄激素受體並生長在含有雄激素的細胞培養基中,能誘導核感受器交互作用蛋白質的表現。染色質免疫沉澱法的實驗結果顯示, 雄激素受體不僅能與核感受器交互作用蛋白質啟動子區域內的雄激素反應元素相結合,也能間接地透過與Sp1的交互作用而束縛在Sp1的結合區。除此之外,雄激素能誘導活化核感受器交互作用蛋白質基因調控區域內包含雄激素反應元素的啟動子以及包含Sp1結合區但雄激素反應元素喪失的啟動子,但在雄激素反應元素與Sp1結合區同時失去的啟動子卻失去活性。這些結果表明,雄激素與其受體能藉著核感受器交互作用蛋白質啟動子區域內的雄激素反應元素與Sp1結合區激活核感受器交互作用蛋白質基因的表現。 用小干擾核糖核酸減少核感受器交互作用蛋白質的表現之後,不論培養基有無雄激素的存在,細胞內雄激素受體的穩定性明顯的降低許多;在相同的情形之下雄激素受體基因的轉錄與核遷移卻不因缺少核感受器交互作用蛋白質的表現而受到任何影響。但是,在培養基內加入MG132抑制劑,細胞內的蛋白質水解酶的活性被抑制之後,降低的雄激素受體蛋白質表現量恢復正常;這個結果說明了,因核感受器交互作用蛋白質的減少而造成雄激素受體蛋白質的降解是透過蛋白質水解酶的活性。核感受器交互作用蛋白質表現量的減少,因雄激素受體蛋白質的降解,更進一步地造成細胞的成長受阻與凋亡。 核感受器交互作用蛋白質會與雄激素受體和Sp1形成複合體,並共同結合在攝護腺特異抗原與自己基因的啟動子區域。核感受器交互作用蛋白質的啟動子活性與基因的轉錄,在雄激素與其受體存在下,會隨著以質體送入的核感受器交互作用蛋白質的量而等比例增加,表示核感受器交互作用蛋白質透過雄激素受體的活性調控它自己的基因表現。簡而言之,核感受器交互作用蛋白質的功能被視為一個雄激素受體輔助活化因子,能穩定雄激素受體蛋白質的表現,並增加雄激素受體的轉錄活性;藉此核感受器交互作用蛋白質不僅受到雄激素受體以及Sp1的活化,並且也能夠自我調控它自己的啟動子活性與基因轉錄。 | zh_TW |
dc.description.abstract | Prostate cancer is the second-leading male-specific death in the world. The androgen-AR signaling has been considered as the primary cause in the prostate cancer progression. AR coactivators play a pivotal role in the AR signaling and are expressed abnormally in some prostate cancer tissues. Androgens act through the AR to target androgen response elements (AREs) in the promoter of AR target genes (e.g.,
p21, FGF8, etc.) which stimulate cell proliferation and ultimately tumor formation. Nuclear receptor interaction protein (NRIP) is recently found in our lab in the yeast-two hybrid system using C-terminus of AR as a bait. NRIP is an AR coactivator which can activate PSA transcription in an androgen-dependent manner. NRIP directly interacts with AR and is involved in cell proliferation and survival in AR-sensitive LNCaP cells, a prostate cancer cell line. Small interfering RNA (siRNA)-mediated knockdown of NRIP expression reduces the transcription of AR target genes and results in cell apoptosis. NRIP expression is increased in AR-regulated LNCaP cells in the presence of androgens. In this thesis, we further investigated the regulation mechanism of NRIP and the role for AR protein stability. We found that NRIP is a novel AR target gene and NRIP plays a role in the AR signaling and the prostate cancer cell line. The core promoter region of NRIP ranges from -413 to +94 upstream the transcription initiation site. The promoter DNA sequences are composed of hormone response elements (ARE, GRE) and three Sp1 binding sites (Sp1-1, Sp1-2, Sp1-3). NRIP is a TATA-less promoter with GC-rich elements which is regulated by Sp1. Transcription of NRIP is increased in the presence of androgens in LNCaP cells in a dose-dependent manner and induced in the AR-deficient 293T cells as AR expression plasmids are transduced. In chromatin immunoprecipitation (ChIP) assays, AR not only associates on the ARE within the NRIP promoter region but also indirectly on Sp1 binding site through interaction with Sp1. In addition, the activity of NRIP is induced by androgens in the ARE-containing promoter as well as the ARE-losing, Sp1 binding site-containing promoter, but not in the ARE and Sp1 double mutated promoter. This result indicates that androgen-AR activates NRIP promoter not only through ARE but Sp1 binding site. Knockdown of NRIP by siRNAs against NRIP gene (siNRIP) reduces the stability of the AR protein regardless of the presence of androgens. However, knockdown of NRIP has no effect on AR transcription and nuclear translocation. The expression of AR protein was restored in the NRIP-knockdowned LNCaP cells which are treated with proteasome inhibitor MG132, demonstrating that knockdown of NRIP contributes to AR degradation through proteasmoe activity. Knockdown of NRIP decreases the AR protein level and consequently results in cell growth arrest and apoptosis. NRIP forms a complex with AR and Sp1 which are recruited to the promoter of AR target genes of PSA and NRIP. The promoter activity and transcription of NRIP gene are increased with the transiently introduced NRIP expression plasmids in a dose-dependent and AR-dependent manner, suggesting NRIP regulates its own promoter through AR activity. Taken together, NRIP acts as an AR coactivator that stabilizes AR protein and enhances AR transcriptional activity in which NRIP autoregulates its own promoter through androgen-mediated AR transactivation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T15:43:30Z (GMT). No. of bitstreams: 1 ntu-97-F91445122-1.pdf: 1814953 bytes, checksum: cab47c53a8fce6cf0260c1545b64331e (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | 口試委員會審定書……………………………………………………i
致謝……………………………………………………………………ii 中文摘要……………………………………………………………iv ABSTRACT………………………………………………………………vi CHAPTER 1. INTRODUCTION……………………………………………1 1.1 Androgen and Androgen Receptor……………………1 1.2 Androgen Response Elements and AR Target Genes…2 1.3 Androgen Receptor Coactivators………………………8 1.4 Nuclear Receptor Interaction Protein……………17 1.5 Specific protein-1 (Sp1) transcription factor…19 1.6 Gene Autoregulation of NRIP and AR Signaling……21 1.7 Prostate Cancer Therapy………………………………22 1.8 Aim of Thesis…………………………………………...24 2. MATERIALS AND METHODS…………………………………29 2.1 Cell Culture and Drug Treatments……………………29 2.2 Plasmid Constructions and Site-directed Mutagenesis……………………………………………………………29 2.3 RNA Interference (RNAi)………………………………32 2.4 Lentivirus Production and Infection………………33 2.5 Transient Transfection and Luciferase Assays……34 2.6 RT-PCR Analysis…………………………………………37 2.7 Co-immunoprecipitation and Western Blot Analysis…………………………………………………………40 2.8 Preparation of Cytoplasmic and Nuclear Extracts……………………………………………………………43 2.9 Chromatin Immunoprecipitation (ChIP)……………44 2.10 Cell Proliferation Assay and Apoptosis Detection………………………………………46 3. RESULTS…………………………………………………49 3.1 Identification of human NRIP gene promoter……49 3.2 NRIP is a novel AR-target gene……………………50 3.3 Sp1 activates NRIP gene expression…………….53 3.4 AR and Sp1 associate on the NRIP promoter and cooperatively regulate NRIP promoter activity…………55 3.5 NRIP positively autoregulates its own gene expression……………………………………………………………59 3.6 NRIP stabilizes AR protein but has no effect on AR mRNA and AR nuclear translocation………………………61 3.7 NRIP acts as a therapeutic target for prostate cancer…………………………………………………………64 4. DISCUSSION……………………………………………67 4.1 Brief summary……………………………………………67 4.2 NRIP is a novel AR target gene……………………69 4.3 Expression of AR coactivator in prostate cancer………………………………………………………………70 4.4 Sp1 regulates the NRIP promoter……………………71 4.5 Androgen induction of NRIP through Sp1 binding site……………………………………………………………………72 4.6 Models of the regulation of NRIP promoter……75 4.7 Autoregulation of NRIP gene and AR signaling……76 4.8 NRIP and AR activity……………………………………78 4.9 Therapeutic target and future prospects…………81 4.10 Conclusion…………………………………………………83 BIBLIOGRAPHY…………………………………………………………85 APPENDIX……………………………………………………………101 | |
dc.language.iso | en | |
dc.title | 雄激素感受器(AR)的共同活化因子,核感受器交互作用蛋白質,由AR和Sp1調控並透過穩定AR 蛋白質而前饋激活它自己的基因 | zh_TW |
dc.title | Nuclear receptor interaction protein, a coactivator of androgen receptors (AR), is regulated by AR and Sp1 to feed forward activate its own gene expression through AR protein stability | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 李明學(Ming-Shyue Lee),張智芬(Zee-Fen Chang),黃麗華(Lih-Hwa Hwang),葉秀慧(Shiou-Hwei Yeh) | |
dc.subject.keyword | 核感受器交互作用蛋白質,雄激素受體,Sp1,攝護腺特異抗原,自我調控, | zh_TW |
dc.subject.keyword | NRIP,AR,Sp1,PSA,autoregulation, | en |
dc.relation.page | 144 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2008-07-04 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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