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研究生：	曾鈺芬 Tseng, Yu-Fen
論文名稱：	Ras正向調控基因RbAp46在細胞株及小鼠致癌過程中所扮演的角色 The role of RbAp46, a Ras up-regulated gene, in tumorigenesis both in vitro and in vivo
指導教授：	劉校生 Liu, Hsiao-Sheng
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2010
畢業學年度：	98
語文別：	英文
論文頁數：	53
中文關鍵詞：	RbAp46 、Ras 、腫瘤新生
外文關鍵詞：	RbAp46, Ras, tumorigenesis
相關次數：	點閱：89 下載：0
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Ras 蛋白質在細胞內參與調控細胞的增生、轉型以及腫瘤細胞之侵犯與轉移。RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) 經由抑制 matrix metalloproteinases (MMPs) 之活性抑制腫瘤細胞之侵犯性轉移和血管新生。有研究指出Ras活化會增強 histone deacetylase 第一型複合物 (HDAC1) 結合在RECK啟動子上的Sp1結合位置以抑制 RECK 表現。Retinoblastoma associated protein 46 (RbAp46) 最早被發現能與 RB 蛋白質結合，並且參與在 HDAC 複合物中，進而能抑制基因之轉錄，而細胞內的 histone 的調控與腫瘤的形成和轉移有關。因此，RbAp46 在腫瘤形成過程中也許扮演重要的角色。我們實驗室之前已證明 RbAp46 是一個受到 Ras 正向調控的基因，並發現 RbAp46 可以和 HDAC1 與 Sp1 形成複合物。本研究證實這複合物可以在細胞內結合到 RECK 啟動子上，若利用反轉錄病毒攜帶特異性小髮夾 RNA ( shRNA) 抑制 Ras 或是 RbAp46 之表現，可使 RECK 的表現增加，並且有效抑制細胞的 MMP9 的活性與侵犯能力。在癌細胞轉移的小鼠模式中，抑制 RbAp46 之表現會減少肺部的腫瘤形成。我們亦證實 Ha-ras 過度表達之細胞 (7-4 Z3) 確實能轉移到肺的組織。另外在 Ha-ras 所誘導的腫瘤新生過程裡，若是增加外源 RbAp46表現則會抑制該細胞的增生，並且於小鼠中抑制腫瘤的形成。我們亦比較膀胱癌的臨床檢體中發現過度表現突變的 Ras 會伴隨 RbAp46 的表現提高，而 RECK 的蛋白量則相對減少。總言之，當 Ras 蛋白質過量表現時，會活化 RbAp46 的表現，透過抑制 RECK 的表現促進腫瘤細胞之移動與侵犯的能力；而在另一方面，又可以透過抑制細胞增生而抑制腫瘤新生之形成。本研究之發現增進我們對 Ras 相關之致癌機制之瞭解。

Ras protein plays important roles in cell proliferation, invasion, and tumor metastasis. RECK (the reversion-inducing cysteine rich protein with Kazal motifs) is a metastasis inhibitor, which suppresses matrix metalloproteinases (MMPs) in the metastatic cascade. It has been reported that Ras induces Sp1 and HDAC1 to form a complex and bind to the RECK promoter to suppress its activity. RbAp46 (retinoblastoma associated protein 46) binds to the RB affinity column and is a component of HDAC complexes which transcriptionally repress gene expression. We previously identified that RbAp46 is a Ras up-regulated gene by suppressive subtractive hybridization PCR. Here, we demonstrated that RbAp46 is a component of the Sp1/HDAC1 complex. We further revealed that this complex bound to the RECK promoter in the cell by chromatin immunoprecipitation assay. Ras suppressing RECK expression through up-regulation of RbAp46 was demonstrated by Ras and RbAp46 shRNA lentivirus. Our data also showed that knockdown of Ras or RbAp46 expression decreased cell invasion by transwell assay and MMP9 secretion by MMP9 assay kit, SensoLyte® 520. Moreover, suppression of RbAp46 in 7-4 cells (a NIH3T3 derivate harboring an inducible Ha-ras oncogene) decreased nodules formation of the lung in the mouse-xenograft model. We also demonstrated that the 7-4-Z3 cells (harboring a β-galactosidase gene) indeed migrated from tumor to the lung tissue. Over-expression of exogenous RbAp46 in 7-4 cells suppressed Ha-rasval12 induced cell proliferation in vitro and tumor formation in vivo. Finally, Over-expression of mutant Ras and RbAp46 associated with low expression of RECK in clinical bladder cancer tissues compared to normal bladder tissues were detected. In summary, we reveal that Ras up-regulates RbAp46, which induces cell invasion through inhibition of RECK expression and suppresses tumor formation through reducing the cell proliferation in tumorigenesis. Our findings increase the knowledge of Ras-related tumorigenesis.

中文摘要………………………………………………………………………….….I
Abstract……………………………………………………………………………..II
致謝…………………………………………………………………………….…………III
Introduction
I. Role of Ras in tumorigenesis………………………………..…………………1
II. RbAp46 and tumorigenesis…………………………………………………….2
III. Overview of RECK protein and its role on tumor metastasis…….……………3
IV. Role of RbAp46 and RECK in Ras-related tumorigenesis…………………….5
Materials and Methods
I. Cell line and cell culture……………………………………………..………...6
II. IPTG induction…………………………………………………………..…….7
III. Transfection……………………………………………………………….…...7
IV. Lentiviral shRNA infection…………………………………………..………..7
V. Western blot analysis…………………………………………………...……...8
VI. Chromatin immunoprecipitation assay………………………………….9
VII. Tranwell invasion assay………………………………………………...10
VIII. Measuring MMP9 activity……………………………………………10
IX. Lentiviral production…………………………………………………..11
X. Experimental metastatic assay in mice………………………………12
XI. Real time PCR…………………………………………………..........13
XII. Tumor embedding, sectioning and X-gal staining……………………….......14
XIII. BrdU staining assay……………………………………………………….14
XIV. MTT assay………………...………………………………………15
XV. Tumor formation……………………………………………………………15
XVI. Immunoprecipitation…………………………………………………………16
XVII. Statistical analysis…………………………………………………………....16
Results
I. RbAp46 was up-regulated by Ha-ras oncogene……………………17
II. Suppression or Ras and RbAp46 increased RECK expression……….....17
III. RbAp46 interacted with HDAC1 as well as Sp1, and bound to the Sp1site in RECK promoter in 7-4 cells………………………………….18
IV. RbAp46 suppressed cell invasion and negative regulated the MMP9 activity…………………………….……………………………….19
V. Suppression of RbAp46 decreased nodule formation and lung weight and in mouse-xenograft model……………………………………………20
VI. Over-expression of exogenous RbAp46 in 7-4 cells decreased tumor formation in mice…………………………………………………………………21
VII. Over-expression of RbAp46 suppressed cell proliferation.............................................22
VIII. A correlation among Ras, RbAp46, and RECK was detected in clinical bladder cancer tissues...............22
IX. RbAp46 interacted with Sp1 was detected by immonoprecipitation in 7-4 cells...........………………………………...23
Discussion…………………………………………………………………………....................24
References……………………………………………………………………......................29
Table and Figure List
Figure 1 The expression of Ras protein correlated with the expression of RbAp46 in NIH3T3 and human T24 cells……......34
Figure 2 Expression level of Ras and RbAp46 protein inversely correlated with RECK protein in bladder cancer T24 cells.................................................35
Figure 3 RbAp46 interacted with HDAC1 as well as Sp1 and bound to the Sp1 site in the RECK promoter in 7-4 cells...36
Figure 4 Silencing Ras or RbAp46 expression decreased invasive activity of bladder cancer T24 cells……...........37
Figure 5 Silencing Ras or RbAp46 expression decreased MMP9 activity of T24 cells..................................………39
Figure 6 RECK expression, lung weight and metastasis of 7-4 Z3 cells in the presence or absence of IPTG together with or without RbAp46 shRNA treatment in nude mice..……….......41
Figure 7 The suppressive effect of exogenic RbAp46 overexpression on Ras-induced tumor formation in SCID mice......................................................44
Figure 8 The suppressive effect of exogenic RbAp46 over-expression on Ras-induced proliferation of 7-4 cells......46
Figure 9 Correlation of the expression of mutant Ras, RbAp46, and RECK proteins in the bladder cancer patient specimens.............................................…………47
Figure 10 Association of RbAp46 with Sp1 increased after IPTG induction of Ras over-expression in 7-4 cells........48
Appendix List
Appendix 1. RbAp46 is up-regulated by Ha-ras oncogene in bladder cancer cell line T1R1.............................49
Appendix 2. The interaction among RbAp46, HDAC1 and Sp1...50
Appendix 3. RbAp46 is involved in three major histone deacetylase (HDAC) complexes: SIN3, NuRD, and PcG complex.51
Appendix 4. Plasmids for lentiviral production............52
                                    

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校內：2011-08-30公開
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