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標題: | 探討TGF-β調控CLIC4基因表現路徑中可能的轉錄因子 Investigation of putative transcription factors involved in TGF-β mediated CLIC4 expression |
作者: | Shin-Nan Lee 李昕南 |
指導教授: | 陳進庭(Chin-Tin Chen) |
關鍵字: | CLIC4,TGF-β,纖維母細胞,成肌纖維細胞,轉錄因子, CLIC4,TGF-β,fibroblast,myofibroblast,transcription factors, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 腫瘤的進程中,腫瘤細胞會誘導周遭其他細胞的轉型,例如使纖維母細胞(fibroblast)轉型為成肌纖維細胞(myofibroblast)。這些轉型的細胞最後會形成腫瘤基質(tumor stroma),提供腫瘤細胞生長所需的環境,使腫瘤進一步的發展。Fibroblast-myofibroblast transition發生的可能機制之一是透過腫瘤細胞分泌轉型生長因子β ( Transforming Growth Factor β, TGF-β) 去調控fibroblast細胞中CLIC4的基因表現,使其上升進而影響細胞週期和轉形,最終被誘發轉形成為myofibroblast的細胞群,可提供有利於腫瘤細胞生存相關的激素及基質,藉由調控微環境因素而促使癌症發生。但由於此路徑中CLIC4基因的上游調控機制尚不明瞭,所以本研究中,利用TGF-β處理fibroblast細胞作為研究模式,希望能藉此找出調控CLIC4基因表現的作用分子。於確立細胞實驗模式之後,分析CLIC4 啟動子區域中可影響基因表現的片段,再經由軟體分析比對出可能對該片段具有調控能力的轉錄因子 (putative transcriptional factors),並以RNA interference的方式進一步確認特定分子與CLIC4之間的關聯性,期望能藉此找出可調控CLIC4基因表現的上游轉錄因子,釐清此基因的調控機制。由報導基因實驗結果中,本研究發現小鼠CLIC4啟動子於(-2000 ~ -1200)的片段上有可能的轉錄抑制者(repressor),而(-234 ~ -56)上則有促進表現的轉錄因子(activator)的結合位置。同時在利用shRNA抑制可能的轉錄因子E2F1與SP1時也發現抑制此二因子時並不會對CLIC4的表現造成影響。未來希望能再將可能與轉錄因子結合的啟動子區域做進一步的縮小範圍,並透過反向染色質免疫共沉澱分析(reverse chromatin immunoprecipitation)找出可能與CLIC4啟動子區域結合的轉錄因子,希望此研究成果對於將來探討惡性腫瘤的進程能夠有進一步的幫助。 Fibroblast-myofibroblast transition plays a crucial role in tumor development. During tumor progression, TGF-β secreted by tumor cells stimulates nearby fibroblast to transform into myofibroblast, which further involves in tumor malignancy by producing growth factors and extracellular matrices. Chloride intracellular channel 4 (CLIC4) protein has been shown involving in the transformation of fibroblasts into myofibroblasts. However, the molecular mechanisms remain unclear. In this study, we intend to address the possible transcription factors involved in TGF-β-mediated CLIC4 expression. To fulfill this goal, we first constructed various reporter constructs with different promoter regions of mouse CLIC4 and measured their activity changes after TGF-β treatment. After confirming the promoter region affected by TGF-β, we further analyzed the DNA sequence to find out the possible transcriptional factors, which were further confirmed by shRNA knockdown. Our results showed that a transcriptional inhibitor binding site was located at -2000 bps to -1200 bps of the mouse CLIC4 promoter (+1 being the transcriptional start site). Meanwhile, we also identified an activator binding site at the range of -234 to -22 of CLIC4 promoter. Further studies showed that the putative transcriptional factors E2F1 and SP1 were not involved in the TGF-β-mediated gene expression of CLIC4. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56596 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
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