金屬感應轉錄因子與缺氧誘導因子之交互作用及對其轉錄活性的影響

MTF-1 interacts with HIF-1α and regulates its transcriptional activity

陳俊豪(CHUN-HAO CHEN)

指導教授 ： 林立元

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2013年)

https://doi.org/10.6843/NTHU.2013.00012

摘要

缺氧誘導因子HIF-1α是腫瘤發展初期很重要的指標性蛋白之一，其會受到腫瘤內部產生之缺氧環境誘導表現且促使血管新生導致腫瘤轉移。金屬感應轉錄因子MTF-1在哺乳類動物胚胎發育扮演一個重要的角色，控制了多數與細胞內鋅離子恆定及抗氧化之相關基因的表現。本篇研究中我們首先利用共同免疫沉澱技術證實了在細胞內兩者蛋白有交互作用的現象發生，利用MTF-1不同片段的質體與HIF-1α共同表現，發現HIF-1α與MTF-1之所有區域皆發生交互作用的現象，且利用HIF-1α之去除ODD區域質體，我們也證實HIF-1α負責蛋白質穩定度之區域並非與MTF-1交互作用主要片段。另外本篇研究發現細胞在鋅離子的刺激下，MTF-1與HIF-1α交互作用會增高，且該交互作用會隨著鋅離子處理劑量逐漸升高而加強，推測可能與HIF-1α受到鋅離子刺激有關，透過西方墨點法我們證實施加鋅離子會導致HIF-1α蛋白表現增加，且利用cycloheximide的處理發現此表現量增加並非透過增強蛋白質穩定度的途徑。接下來我們利用分離細胞質與細胞核萃取液的方法探討在MTF-1存在是否會影響HIF-1α的細胞質與細胞核的分佈，研究結果顯示MTF-1的存在並沒有顯著影響HIF-1α的分佈，然而利用報導基因活性檢測技術及siRNA降低基因表現程度發現MTF-1的存在使HIF-1α轉錄活性顯著提升。

關鍵字

金屬感應轉錄因子 ； 缺氧誘導因子 ； 鋅

並列摘要

Hypoxia-inducible factor- 1α (HIF-1α) is an important protein involved in tumor development. Production of HIF-1α would be stimulated by the hypoxic environment in tumor and leads to tumor metastasis. Metal responsive transcription factor 1 (MTF-1), which plays an essential role in modulating the expression of genes, involves in cellular zinc homeostasis. In this study, we investigated the interaction between HIF-1α and MTF-1 by immunoprecipitation. The results revealed that HIF-1α interacts with all of the domains of MTF-1. Besides, we also expressed MTF-1 and HIF-1α with the deletion of ODD domain. The results showed that loss of ODD domain, a region responsible for protein stability, had no effect on HIF-1α and MTF-1 interaction. Noticeably, addition of zinc to cells increased MTF-1/ HIF-1α interaction in a dose-dependent manner. According to the results, addition of zinc increased the HIF-1α protein level. However, this increase was not due to the alternation of its mRNA level by enhancing protein stability through the use of cycloheximide. Analyzing the position of HIF-1α in cytoplasmic and nuclear fraction showed that the presence of MTF-1 did not affect the distribution of HIF-1α. Using reporter gene assay and siRNA to reduce the expression of MTF-1 mRNA revealed that the presence of MTF-1 would increase the transcriptional activity of HIF-1α significantly.

並列關鍵字

MTF-1 ； HIF-1α ； Zinc

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