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標題: | 氯離子通道蛋白CLIC4基因之調控機轉探討 The Regulatory Mechanisms Involved in CLIC4 Gene Expression |
作者: | Mu-Ching Huang 黃睦晴 |
指導教授: | 陳進庭 |
關鍵字: | myofibroblast,TGF-β,啟動子,轉錄因子,G-quadruplex,甲基化,CRISPR/Cas9, myofibroblast,TGF-β,promoter,transcription factor,G-quadruplex,DNA methylation,CRISPR/Cas9, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | 在腫瘤發展的過程中,CLIC4基因在腫瘤細胞中的表現逐漸減少,而在腫瘤微環境中的基質細胞表現卻逐漸增加,在腫瘤細胞及基質間對於CLIC4表現的差異性,和腫瘤的發展歷程有一定的關連性,且對於腫瘤發展皆有正向的影響。然而目前關於此基因的表現是如何被調控,所知卻相當有限,因此本研究主要的目的為探討CLIC4基因相關的調控機制。已知腫瘤微環境中所富含的fibroblast,主要受到癌細胞分泌之TGF-β誘導而轉變為myofibroblast。藉由MRC5細胞的研究結果顯示,CLIC4的轉錄在此過程中會受到TGF-β所誘導而上升,同時,CLIC4也能進一步調控下游分子MMP9的表現。分析CLIC4啟動子(promoter),發現當中有轉錄因子、潛在的二級結構以及甲基化修飾共同存在的區域。透過SEAP reporter assay分析找出潛在的轉錄因子結合的區域;之後藉由軟體預測CLIC4啟動子中含有形成G-quadruplex (G4)二級結構的序列,並利用NMR分析幾段具有較高潛力形成G4的片段(Potential G4s,PG4s),證實這些序列確實能夠形成G4結構。reporter assay分析結果顯示,當中的PG4-3結構對於CLIC4轉錄可能是重要的,進一步以CRISPR/Cas9直接編輯genome中CLIC4 啟動子序列,透過pop-in / pop-out策略挑選破壞genome中PG4-3序列的cell clone,在in vivo中證實此G4結構對於CLIC4表現的重要性,目前推測PG4-3可能作為阻礙repressor結合的角色;最後,我們觀察到甲基化修飾的現象確實存在於腫瘤細胞中,且與CLIC4表現量有相關性,說明甲基化修飾可能是腫瘤細胞調控CLIC4表現的方式。綜合以上的研究,我們證實CLIC4 啟動子中的二級結構-G4,以及甲基化修飾,對於CLIC4基因表現調控扮演著重要的角色。 During tumor progression, chloride intracellular channel protein 4 (CLIC4) has been found to be downregulated in tumor cells but upregulated in stroma cells in tumor microenvironment. The reciprocal expression of CLIC4 in each site could contribute to cancer malignancy. However, little is known about how this gene is regulated. In this study, we investigated the regulatory mechanisms involved in CLIC4 gene transcription. It has been shown that fibroblast, one of the major components of stromal cells is activated by TGF-β and become myofibroblast. During this process, the transcription of CLIC4 is also induced by TGF-β; meanwhile, the expression of MMP9 is mediated by CLIC4. Upon dissecting CLIC4 promoter, putative transcription factor binding sites, the possible secondary structure and DNA methylation were found in common region. Reporter assay was conducted to defined critical region. G-quadruplex (G4) forming sequences were predicted and could actually form G4 structure in vitro with NMR. The reporter assay showed that G4 structure might have their significance in CLIC4 expression. Using CRISPR/Cas9 system to edit CLIC4 promoter sequence in cell genome mediated by pop-in/pop-out strategy, we also confirmed the significance of G4 structure for CLIC4 expression in vivo. Moreover, DNA methylation of CLIC4 promoter was found to be correlated with its mRNA expression level, implying DNA methylation might be one of the regulatory mechanisms of cancer cell to downregulate CLIC4 expression. In conclusion, we’ve found the secondary structure-G4 and methylation in CLIC4 promoter play a role in transcriptional regulation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79163 |
DOI: | 10.6342/NTU201802204 |
全文授權: | 有償授權 |
電子全文公開日期: | 2023-08-17 |
顯示於系所單位: | 生化科技學系 |
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