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系統識別號 U0026-1208201521031000
論文名稱(中文) 肺癌中核內前胸腺素表現抑制轉化生長因子-β引起的上皮細胞間質轉化
論文名稱(英文) Nuclear prothymosin α suppresses TGF-β-induced epithelial-mesenchymal transition in lung cancer
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 103
學期 2
出版年 104
研究生(中文) 陳藜云
研究生(英文) Li-Yun Chen
學號 S16021125
學位類別 碩士
語文別 英文
論文頁數 54頁
口試委員 指導教授-吳昭良
口試委員-蕭璦莉
口試委員-林季千
口試委員-張孟雅
口試委員-蘇五洲
中文關鍵字 前胸腺素  肺癌  Smad7 
英文關鍵字 Prothymosin α  Lung cancer  Smad7 
學科別分類
中文摘要 乙型轉化生長因子 (transforming growth factor-β, TGF-β) 參與及調控體內許多複雜的訊息傳遞網路,而TGF-β也參與在癌症的進展過程。在腫瘤發生的前期,TGF-β會藉由誘導細胞週期停滯以及引發細胞凋亡,扮演抑制腫瘤生長的角色;然而,在腫瘤生長的後期,TGF-β卻扮演著促進腫瘤生長的角色,藉由調控增強癌細胞的移動及侵犯能力,誘導上皮-間質轉化 (epithelial-mesenchymal transition, EMT)的發生。EMT是造成腫瘤進行遠端轉移的重要機制,癌細胞會和原腫瘤脫離細胞間的連結,由原本的上皮細胞轉換成間質細胞,造成轉移的發生。前胸腺素 (prothymosin α, ProT) 是酸性核蛋白,會調節細胞的轉錄、染色質重建以及免疫調節等。在臨床肺癌腫瘤切片中,利用免疫染色我們發現,在第一、二期癌症病患腫瘤中,ProT會大量表現在細胞核,而隨著癌症的進展,在後期細胞核內ProT的表達量明顯減少,甚至消失。因此我們推測細胞核內的ProT可能參與在TGF-β誘導EMT造成轉移發生的過程中,我們發現ProT會抑制TGF-β所促使的Snail, Twist, N-cadherin表達上升的現象,並且回升E-cadherin的表現。而我們認為ProT抑制TGF-β的訊息傳遞是藉由調控Smad7的表現及功能,ProT會促使Smad7 乙醯化,增強Smad7的穩定性,進而促使Smad7在細胞核中競爭Smad2在Snail啟動子上的結合位置,干擾了TGF-β誘導Snail的表達而抑制EMT。在小鼠實驗中,我們也證實ProT高量表達的人類肺癌A549細胞,小鼠背上腫瘤轉移到肺部的情形明顯較弱於對照組。分析臨床肺癌腫瘤的切片,發現細胞核內ProT的表達情形和Snail的表達,在癌症進展的過程中明顯呈現負相關。總結以上,在本研究中我們指出,細胞核內的ProT會藉由促使Smad7乙醯化進而調控Smad7作為轉錄抑制的角色,抑制Snail的表達,而抑制了TGF-β誘導EMT的發生。
英文摘要 The multifunctional cytokine transforming growth factor β (TGF-β) regulates a complicated signaling network. In cancer progression, TGF-β exerts its tumor-suppressive role by inducing cell-cycle arrest and apoptosis at early stages. Nevertheless, at the late stages of cancer, TGF-β promotes tumor progression by enhancing migration and invasion to induce epithelial-mesenchymal transition (EMT). EMT has been shown to be a critical mechanism of tumor cell metastatic dissemination by endowing cells with a more motile, invasive potential. Prothymosin α (ProT) is an acidic nuclear protein and an important regulator of cell proliferation, transcription, chromatin remodeling, and immunomodulation. We found immunohistochemically that ProT expression was associated with cancer progression. At the early stage, ProT was overexpressed in the nucleus and during disease progression ProT expression was decreased and its nuclear expression was even lost. Therefore, we hypothesized that nuclear ProT may play a pivotal role in interrupting TGF-β signaling, resulting in inducing EMT. We found that TGF-β-induced downregulation of E-cadherin and upregulation of Snail and N-cadherin could be supressed by ProT overexpression in A549 cells. Moreover, we demonstrated that ProT could antagonize TGF-β signaling through enhancing Smad7 acetylation and stability. We further showed that Smad7 disrupted the binding of Smad2 to the promoter of Snail1, a TGF-β-induced EMT-associated regulator, leading to downregulating Snail1 expression. In addition, we found that human lung cancer A549 cells overexpressing PoT exhibited lower metastatic potential than parental A549 cells in NOD/SCID mice. In human lung cancer specimens, there was an inverse correlation between nuclear ProT and Snail expression. In conclusion, nuclear ProT expression enhances Smad7 acetylation to antagonize TGF-β signaling, which competes the Smad2-binding site on the Snail1 promoter to reduce Snail1 expression and inhibit TGF-β-induced EMT.
論文目次 Astract.................................................I
Chinese Abstract...............................................II
Achknowledgement......................................III
Content................................................IV
Figure content........................................VII
Introduction............................................1
I. Transforming growth factor β signaling (TGF-β).......1
I.1 TGF-β...............................................1
I.2 Role of TGF-β in cancer progression…………………………………….1
I.3 TGF-β signaling and Smads family……………………………………….2
II. Epithelial-mesenchymal transition (EMT)…………………………….3
III. Prothymosin α (ProT)………………………………………………………3
III.1. Structure and biological roles of ProT……………………………………….3
III.2. Role of ProT in regulation of acetylation……………………….……………4
Aim………………………………………………………………………………….5
Materials and methods……..………………………………………………………6
Materials………………………………………………………………………...6
I. Plasmids……………………………………………………………………….6
I.1. Expression vectors……………………………………………………………6
I.2. Short hairpin RNA (shRNA)………………………………………..………6
II. Cell lines…………………………………………………….…………..……6
III. Primers………………………………………………………………………7
IV. Antibodies……………………………………………………………….……8
IV.1. Primary antibodies…………………………………………………..………8
IV.2. Secondary antibodies……………………………………………….………9
V. Animal model……………………………………………………….………9
VI. Bacteria.…………………………………………….………………………...9
VII. Recombinant protein...……………………………………………….………9
VIII. Reagents……………………………………….…………………..………10
VIII.1. Bacterial growth media…………………………………….……………10
VIII.2. Cell lysis buffers…...……………………………………….……………11
VIII.3. Immunoblotting buffers…………………………………….……………12
Methods………………………………………………………………………...13
Cell cultures and treatments…………………...………………………………...13
Cell transfection……………………………….………………………………...13
Lentivirus production….………………………………………………………...13
Transwell assay…….….………………………………………………………...14
Wound healing assay.….………………………………………………………...15
RNA isolation and reverse transcription………………………………………....15
Real-time quantitative PT-PCR analysis………………………………….......15
Immunoblotting………………………………………………………................16
Immunoprecipitation ………………………………………………………........16
Chromatin immunoprecipitation (ChIP)…………………………..…………….17
Immunofluorescence staining……………………………….………………......17
Histological and immunohistochemical staining………………………………...18
In vivo tumor metastasis analysis……………...………………………………...18
Clinical specimens……………………...………………………….……….…...19
Statistical analysis………………………..……………………………………...19
Results………………………..……………………………………………………20
Nuclear ProT expression is decreased in lung cancer of late-stage patients.…..…20
High expression of ProT inhibits expression of EMT-associated transcription factors by interrupting TGF-β signaling……………………...………....……….20
ProT antagonizes TGF-β signaling through the regulation of Smad7 ………...…21
ProT promotes Smad7 acetylation to stabilize Smad7 expression ………………23
ProT regulates Smad7-mediated transcriptional repression of the Snail1 gene by enhancing Smad7 acetylation and sequestering it in the nucleus………...………24
Nuclear ProT expression suppresses metastatic growth of human lung tumor xenografts………….……….………………………………………………...…25
Nuclear ProT expression in human lung cancer specimens is associated with tumor pathologic features………………………………………………………………26
Discussion……………………..………………………………….………………28
Conclusion……………………..…………………………………………………31
References……………………..…………………………………………………32
Figure legends………………..……………………………………………………37
Appendix………………..…………………………………………………………51
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