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研究生:賴宛廷
研究生(外文):Wan-Ting Lai
論文名稱:低氧誘導轉錄輔因子(Cited2)促進低氧誘發因子(HIF-2 alpha)調控肺癌細胞生長之研究
論文名稱(外文):Cited2,a hypoxia inducible transcriptional coactivator, promotes HIF-2 alpha mediated cell proliferation of lung cancer
指導教授:黃銓珍吳成文
指導教授(外文):Chang-Jen HuangCheng-Wen Wu
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:64
中文關鍵詞:缺氧誘發因子肺癌細胞轉錄輔因子
外文關鍵詞:hypoxia inducible factorlung cancertranscriptional coactivator
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雖然已知低氧環境是影響腫瘤生長的重要因素,低氧的壓力會促進腫瘤的增殖,但探討低氧的壓力是如何調控癌細胞增生的研究卻不多。本篇實驗主要是探討轉錄輔因子,Cited2,不但會因低氧的刺激而被誘導,更能夠肺癌細胞在低氧的環境下下生長。根據西方點墨法(Western blot)及即時定量聚合酶連鎖反應(Q-PCR)結果顯示,過量表達低氧誘發因子(HIF-2alpha)會誘導Cited2的表達;若降低低氧誘發因子(HIF-2alpha)的表達,因低氧所誘導表現的Cited2也會降低其表現量;以上結果說明低氧所誘發的Cited2表現,是藉由低氧誘發因子(HIF-2alpha)所調控,而非藉由低氧誘發因子(HIF-1alpha)。鑒於在癌細胞中高量表達低氧誘發因子(HIF-2alpha),會促使細胞增殖及細胞不貼附生長的特性的同時,將細胞中的cited2表現量降低,便抑制了由低氧所調控的癌化能力,更說明了在低氧誘發因子(HIF-2alpha)所調控癌化特性上,Cited2扮演一個的角色。如同我們在細胞週期的實驗中所觀察到的,低氧的壓力造成細胞停滯在細胞週期中的G1/S期,進而若將Cited2的表達量降低,會大幅提升由低氧壓力所造成的細胞G1/S停滯之比率。次外,過量表達Cited2,也可以促使細胞在鐵離子螯合劑的處理之下生長。根據以上的實驗結果,我們證實Cited2的確調控了細胞的低氧誘發之生長。轉錄因子E2F3在細胞週期中,是使細胞從G1期進入S期的主要調控者,而在微陣列技術分析中已證實Cited2調控E2F3。從西方點墨法及即時定量聚合酶連鎖反應結果中,證實低氧的刺激以及過量表達低氧誘發因子(HIF-2alpha),都會誘導E2F3的表現,顯示E2F3為低氧誘發基因。在缺乏Cited2表達的肺癌細胞中,E2F3的表現量也隨之降低,顯示Cited2調控了低氧誘發的E2F3表現量。已知低氧誘發因子(HIF-2alpha)藉由增加c-Myc的活性進而促使細胞的生長。免疫沉澱分析結果顯示Cited2會和低氧誘發因子(HIF-2alpha)及c-Myc互相結合,並增加低氧誘發因子(HIF-2alpha)/c-Myc的轉錄活性。在本次篇實驗中,我們假設在低氧誘發因子(HIF-2alpha)誘導Cited2的同時,進而促使了低氧誘發因子(HIF-2alpha)/c-Myc的轉錄活性,使得細胞在低氧的壓力下得以繼續增殖。
Though it is known that hypoxic microenvironments in solid tumor shape tumor progression, little is understood about how hypoxia regulates cancer cell proliferation. We have identified that Cited2, a novel transcriptional coactivator, is induced by hypoxia, and regulates hypoxic cell cycle progression in lung cancer. Immunoblotting and Q-PCR analysis showed that overexpression of HIF-2α but not HIF-1α promoted Cited2 expression. Knockdown of HIF-2α but not HIF-1αattenuated hypoxia-induced Cited2 level, suggesting that hypoxia induces Cited2 expression through a HIF-2α dependent pathway. Whereas overexpression of HIF-2α promoted cell proliferation as well as anchorage–independent cell growth, knockdown of Cited2 inhibited HIF-2α-mediated oncogenesis of lung cancer, suggesting that hypoxia-induced Cited2 is important for HIF-2α-mediated oncogenic properties. Consistent with these observations, cell cycle analysis showed that hypoxia induced G0/G1 cell arrest, and knockdown of Cited2 further increased hypoxia-mediated cell cycle arrest. In addition, overexpression of Cited2 promoted cell growth under the treatment of hypoxia-mimetic agents. These data support the notion that Cited2 regulates hypoxia-mediated cell cycle progression. Microarray analysis identified that Cited2 controlled E2F3, a key factor regulating G0/G1 cell cycle transition. Immunoblotting and Q-PCR analysis demonstrated that both hypoxia and HIF-2αinduced E2F3 expression, indicating E2F3 as a hypoxia inducible genes. Cited2-silencing attenuated hypoxia-induced E2F3 expression in lung cancer cells, suggesting that Cited2 regulates hypoxia-mediated E2F3 expression. It has been shown that HIF-2αpromotes cell proliferation through enhancing c-Myc transcription. Coimmunoprecipitation indicated that Cited2 interacted with HIF-2αand c-Myc. E2F3 promoter reporter analysis further showed that Cited2 enhanced HIF-2α/c-Myc-mediated transactivation of E2F3 promoter. Thus, our study provides a novel mechanism that shows how HIF-2α induces Cited2 expression, which further promotes HIF-2α c-Myc-mediated transcription in a feed-forward manner, leading to increased cell proliferation.
中文摘要(Chinese abstract )---------------------------------4
Abstract---------------------------------------------------6
Introduction
1.1 Cited2-------------------------------------------------8
1.2 Hypoxia-----------------------------------------------10
1.3 Hypoxia inducible factor------------------------------10
1.4 Desferrioxamine --------------------------------------13
1.5 Effects of hypoxia on cell death, proliferation and drug resistance ------------------------------------------13
1.6 E2F3 and cell cycle progression-----------------------14
1.7 c-Myc ------------------------------------------------15
1.8 HIF-2αenhances c-Myc transcription--------------------15
Material and Method
2.1 Cell culture -----------------------------------------17
2.2 Exposure to Hypoxia-----------------------------------17
2.3 Protein extraction and Western blotting---------------18
2.4 Co-Immunoprecipitation and Western Blot Analysis------20
2.5 Lentiviral Infection----------------------------------20
2.6 RNA extraction and RT-PCR-----------------------------21
2.7 Quantitative Real-Time PCR----------------------------22
2.8 Colony formation assay (anchorage-dependent)----------23
2.9 Anchorage-independent assay---------------------------24
2.10 Flow cytometry---------------------------------------25
2.11 Luciferase reporter assay----------------------------25
2.12 WST-1------------------------------------------------26
2.13 Apoptosis cells detection----------------------------26
Result
3.1 Hypoxia via HIF-2 induces Cited2 expression in lung cancer----------------------------------------------------28
3.2 Cited2 regulates hypoxia-mediated cell growth of lung cancer----------------------------------------------------29
3.3 Cited2 promotes HIF-2 alpha/c-Myc mediated E2F3.------31
Discussion------------------------------------------------33
References------------------------------------------------36
Figures
Figure 1 -------------------------------------------------41
Figure 2 -------------------------------------------------43
Figure 3 -------------------------------------------------45
Figure 4--------------------------------------------------47
Figure 5 -------------------------------------------------50
Figure 6 -------------------------------------------------52
Figure 7 -------------------------------------------------54
Figure 8 -------------------------------------------------56
Figure 9 -------------------------------------------------58
Figure 10 ------------------------------------------------60



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