臺灣博碩士論文加值系統

Rad23 是個從人類到酵母菌等高度保留的蛋白。根據目前研究，在酵母菌中的Rad23 會在人類細胞中的分成兩種同源蛋白，hHR23A 和 hHR23B，而目前他們被認為參與蛋白的降解及 DNA 的修復。泛素-蛋白酶體系統(Ubiquitin-Proteasome System, UPS)是一個監控蛋白質品質的系統。當泛素(Ubiquitin)被泛素活化酶(Ubiquitin-activating enzyme, E1) 所 活 化 而 使 泛 素 轉 至 泛 素 結 合 酶
(Ubiquitin-conjugating enzyme, E2)，最後在泛素連接酶(Ubiquitin-ligase, E3)的幫助下將泛素接上欲降解的蛋白。透過這個過程，被接上泛素的蛋白會送至蛋白酶體降解，其中，Rad23 可能參與其中。在本篇論文我們探討了 hHR23A 在人類細胞中可能扮演的角色。首先我們將 hHR23A 的表現量減弱，發現細胞的型態改變且會促使細胞有上皮-間質細胞型態的轉化(epithelial–mesenchymal transition, EMT)現象發生，除此之外我們也發現透過 hHR23A 的表現量減弱，Twist1(Twist-related protein 1)的表現量會上升。透過免疫沉澱法，我們發現 hHR23A 會與 Twist1 有互相結合的現象且調控 Twist1 的穩定性。最後，我們發現當減弱 hHR23A 的表現量時 會使細胞有遷移(migration)及侵襲(invasion)的能力增加 因此我們認為 hHR23A，。
可能扮演調控細胞 EMT 的角色。

Rad23 is an approximately 400 amino acids protein and evolutionarily conserved from yeast to human. Yeast Rad23 and mammalian homologs, hHR23A and hHR23B are originally identified as an important factor involved in DNA repair and targeting ubiquitinated proteins for 26S protein degradation. The Ubiquitin-Proteasome System(UPS) is responsible for protein quality control. Ubiquitin is activated by Ubiquitin-activating enzyme (E1) and transfers to Ubiquitin-conjugating enzyme (E2),the Ubiquitin-ligase (E3) will replace ubiquitin from E2 to target protein and form a polyubiquitin chain. It has been found that hHR23 contains multiple functions. Here, we found knockdown of hHR23A makes the cell morphology change and increases the
expression of Twist1.
Twist1 is a transcription factor that promotes epithelial–mesenchymal transition (EMT). By immuonprecipitation, we found hHR23A interacts with Twist1 and regulates Twist1 protein stability. In addition, we found that depletion of hHR23A caused cell migration and metastasis. Taken together, we showed that hHR23A may have a role to regulate migration, even metastasis.

中文摘要 ….……………………………………………………………………..…….i
Abstract …..………………………………………………………………………..…....ii
Chapter 1. Research background and aims………………………………………….1
The Ubiquitin-Proteasome System (UPS) ….………………..…………………….....1
The Role of Rad23 in Protein degradation..…………………..……………………....2
The role of hHR23 in DNA repair…………………………………………………….4
The regulation of epithelial–mesenchymal transition ..……………………..………...5
The role of Twist in EMT……………………………………………………………..7
Specific aims…………………………………………………………………………..7
Chapter 2. Materials and Methods……………………………………………………9
Cell culture...…………………………………………………………………………..9
Chemicals and Antibodies…………………………………………………………….9
Transfection and Knockdown…………………….………………………………….10
Quantitative real-time PCR…………………………………………..........................10
Preparation of cell lysates….………………………………………...........................11
Immunopricipitation and Western Blot………………………………………………11
Cell migration and invasion assay………………………………………...................12
Statistics………………………………………………...............................................12
Chapter 3. Results……………………………………………………………………..13
Knockdown of hHR23A makes the cell morphology change and increases the
expression of Twist1 in A549 cells…………………………………………………..13
Knockdown of hHR23A increases the expression of Twist1 and mesenchymal
markers in CL1-0 cells………………………………………………………………14
iii

hHR23A regulates Twist1 stability…………………………………………………..14
Knockdown of hHR23A enhance the ability of migration…………………………..15
Knockdown of hHR23A enhance the ability of invasion……………………………15
Chapter 4. Discussion ………………………………...……………………………....16
References ……………………………………………………………………………..18
Figures ………………………………………………………………………………...23

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