臺灣博碩士論文加值系統

　　Ras可以表現出許多致癌基因的特性，包括造成細胞增生，抑制細胞凋亡和促進腫瘤的血管新生和轉移。視網膜細胞瘤(Rb)抑癌基因第46相關蛋白質(RbAp46)首先在Hela細胞中被鑑定出來，可以與Rb親和力管柱結合。以前的研究顯示RbAp46為可以和組織蛋白結合的次單位，參與染色質的重組，像是組織蛋白乙醯化或去乙醯化酵素都可藉其與組織蛋白結合。一些研究顯示Ras可以促進組織蛋白去乙醯(HDACs)結合Sp1的是經由細胞外信號傳遞激(ERK)路徑。我們的實驗室已經證明RbAp46的大量表現也能促進HDAC1 和Sp1之間的結合。本研究發現Ras能促使 RbAp46表現上升。進一步研究顯示Ras調控 RbAp46的信息傳遞路徑是透過Raf/MEK路徑啟動RbAp46啟動子，並且是透過RbAp46啟動子上-290 和-239之間的轉錄因子結合位。另一個基因， 在Bcl-2家族裡屬於僅有BH3的蛋白質的BNIP3。之前BNIP3已經被報告參與細胞之自體吞噬和細胞之死亡路徑中。前人發現Ras能夠促進BNIP3表現，本實驗室以BNIP3啟動子冷光報導基因活性確認，並進一步證明此一活化作用是經由MAPK路徑來進行。而且我們也在具有可調控Ha-rasV12的細胞株中證明Ras能引起LC3-GFP 聚集及自體吞噬作用。同時也以BNIP3 的siRNA證明了Ras是透過促進 BNIP3表現而引起細胞的自體吞噬作用。

　Ras shows oncogenic characteristics toward the biological functions, including induction of the cell proliferation, inhibition of apoptosis, and facilitating angiogenesis and metastasis of tumors. The retinoblastoma (Rb) suppressor associated protein 46 (RbAp46) was first identified as a protein in HeLa cells that binds to an Rb affinity column. Previous studies showed that RbAp46 functions as a core-histone-binding subunit that targets chromatin assembly factors, chromatin-remodeling factors, histone acetyltransferase and deacetylase to their histone substrates in chromatin. Some studies showed that Ras activation induces the binding of histone deacetylases (HDACs) to Sp1 to suppress the RECK expression via extracellular signal-regulated kinase (ERK). Our laboratory has demonstrated that overexpression of RbAp46 could enhance the binding between HDAC1 and Sp1. In this study, we showed that Ras could upregulate RbAp46 promoter. We further studied the signaling pathway utilized by Ras to regulates RbAp46 by analyzing RbAp46 promoter activity. Our data showed that Ras may activate RbAp46 promoter through Raf/MEK pathway which is also required for inhibition of RECK expression. Ras upregulates RbAp46 promoter activity is through TA binding sites between -290 and -239 in the RbAp46 promoter. BNIP3, a BH3-only protein in the Bcl-2 family, has been reported to play a role in autophagy, as well as cell death pathway. We found that ras oncogene upregulates the BNIP3 expression analyzed by BNIP3 promoter assay and Western blotting. Further study demonstrated that MAPK pathway is required. Moreover, we demonstrated that Ras induces the LC3-GFP aggregation and autophagy in two stable cell lines harboring an inducible Ha-rasV12 in a time-dependent manner. In contrast, suppression of BNIP3 by BNIP3 siRNA blocked the autoaphgy induced by Ras, suggesting that Ras is through upregulating BNIP3 to induce autophagy in the cells.

Abstract i
中文摘要 ii
誌謝 iii
Contents iv
List of Figures vi

Chapter 1 Introduction 1

Chapter 2 Materials and Methods
1. Cell lines and cell culture 6
2. Cloning of full length and deletion mutants of RbAp46 promoter 6
3. Plasmid preparation 7
4. Calsium-phosphate transfection 8
5. Promoter activity assay 9
6. Cloning of FLAG tagged BNIP3 and inducible BNIP3 expression system 10
7. Western blotting 11
8. Immunofluorescence assay 11

Chapter 3 Results
Ras and RbAp46
I. Relationship between Ras and RbAp46 promoter
promoter
1. Cloning of RbAp46 promoter 13
2. Ras could upregulate RbAp46 promoter 13
II. Ras downstream pathway Raf/MEK/ERK upregulates RbAp46
1. Ras upregulates RbAp46 promoter via Raf/MEK/ERK pthway 14
III. -500/+1 region of the RbAp46 promoter is upregulated by Ras overexpression
1. The -525/+1 region of RbAp46 promoter is responsible for Ras upregulation 15
2. Ras upregulates RbAp46 promoter within -290/-239 region 16
Ras and BNIP3
I. Ras can induce autophagy of the fibroblast NIH-3T3 cells 18
II. BNIP3 can induce autophagy in NIH-3T3 fibroblast cells
1. Construction of the plasmid continuously expressing BNIP3 19
2. Construction of inducible FLAG tagged BNIP3 expression system 20
3. Overexpression of BNIP3 induced autophagy in NIH-3T3 fibroblasts 20
III. Ras can upregulate BNIP3 expression through Ras/Raf/MEK/ERK pathway 20
IV. BNIP3 involves in Ras induced autophagy 21

Chapter 4 Discussion 22

References 24

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