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標題: | 探討低氧誘導之 miR-103/107 藉由 KLF4 對細胞移動之影響 The hypoxia-induced miR-103/107 targets KLF4 to regulate cell migration |
作者: | Hsiang-Ching Chung 鍾向晴 |
指導教授: | 陳瑞華(Ruey-Hwa Chen) |
關鍵字: | 微型核醣核酸,KLF4, microRNA,KLF4, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 低氧常見於固體型腫瘤,是一個對細胞或組織氧氣供給不正常的現象。當腫瘤細胞處於低氧情況時, 常引起各種反應以因應此狀況,例如血管新生或腫瘤轉移,而調控這些低氧反應最主要的因子是缺氧誘導因子–1 (HIF-1)。近年來,微型核醣核酸 (microRNA) 被發現能受低氧狀況所誘導而增加表現量,例如 miR-103/107 就是其中之一。然而這些微型核醣核酸受低氧誘導表現量增加的機制以及生物功能並不清楚。在本論文中,我們主要探討受低氧誘導表現量增加的 miR-103/107 在細胞移動所扮演的角色。透過冷光酵素活性的測定,我們發現缺氧誘導因子–1 能直接調控 miR-103-1 與 miR-107 的啟動子。除此之外,我們也證實了 miR-103/107 能促進細胞移動。更重要的是,利用 miRNA 海綿 (miRNA sponge) 抑制 miR-103/107 的功能,能阻礙受低氧誘導增加表現之 miR-103/107 促進細胞移動的能力,顯示被缺氧誘導因子–1 引起表現的 miR-103/107 能調控細胞受低氧所促進的移動能力。為了解 miR-103/107 是如何影響細胞移動,我們搜尋了具有抑制細胞移動能力之 miR-103/107 的標靶基因。透過生物信息學預測以及文獻回顧,我們聚焦於 KLF4,它被報導是一個腫瘤抑制因子且具有抑制細胞移動的能力。我們透過實驗證實在多株細胞中, miR-103/107 能標靶 KLF4 信使 RNA (mRNA) 的 3 端非編碼區 (3’-UTR) 而抑制內生性 KLF4 蛋白。此外,KLF4 的表現受低氧狀況所抑制,而這個抑制的現象能被反義之 miR-103/107 (anti-miR-103/107) 恢復,暗示了 miR-103/107 在低氧情況下抑制 KLF4 的重要性。有趣的是,當過度表現 KLF4 能抑制 miR-103/107 所促進的細胞移動能力,而剔除 KLF4 則能恢復低氧狀況下 miR-103/107 海綿所抑制的細胞移動能力。這些結果支持了我們提出的概念: miR-103/107 能藉由抑制 KLF4 以促進細胞移動能力。總結來說,我們發現在低氧狀況下,缺氧誘導因子–1 能引起 miR-103/107 表現,而此時 miR-103/107 能透過 KLF4 去促進細胞移動能力。這個新的路徑代表了其中一個低氧促進細胞移動的機制,而且可能參與了癌化的過程。 Hypoxia, a phenomena of abnormally decreased oxygen supply to cells or tissues, is commonly seen in solid tumors. During hypoxia, tumor cells elicit variety of cellular responses such as angiogenesis and metastasis to compromise the hypoxic condition, and the key factor in these processes is hypoxia-inducible factor 1 (HIF-1). Recent studies have shown that miR-103/107 are upregulated by hypoxia. However, the molecular mechanism underlying their upregulation and the biological functions of these miRNAs in hypoxia are still unknown. In this study, we focus on the role of hypoxia-inducible miR-103/107 in cell migration. First, using luciferase activity assay and ChIP assay, we demonstrated a direct regulation of the promoters of miR-103-1 and miR-107 by HIF-1α. We further showed that miR-103/107 promoted cell migration. More importantly, blockage of the action of miR-103/107 by miR-103/107-specific sponge inhibited hypoxia-induced cell migration. These data indicate that HIF-1α-induced miR-103/107 expression participates in hypoxia-induced cell migration. Next, we searched for miR-103/107 targets that mediate the inhibition of migration. Through bioinformatic prediction and literature review, we focused on KLF4, a known tumor suppressor gene involved in the inhibition of cell migration. Experimental validation substantiated that miR-103/107 targeted 3′-UTR of KLF4 to downregulate the endogenous KLF4 expression in multiple cells lines. Moreover, hypoxia-induced KLF4 downregulation was rescued by anti-miR-103/107, indicating an important role of miR-103/107 in KLF4 downregulation in hypoxia. Interestingly, overexpression of KLF4 attenuated miR-103/107-induced cell migration, and shRNA-based downregulation of KLF4 restored the inhibitory effect of miR-103/107 sponge on cell migration during hypoxia. These results support the notion that miR-103/107 promotes cell migration by targeting KLF4. In conclusion, we identify a novel pathway through which HIF-1α-induced miR-103/107 target KLF4 to potentiate cell migration. This pathway represents one mechanism underlying hypoxia-induced motility of tumor cells and may participate in tumor progression. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23791 |
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顯示於系所單位: | 生化科學研究所 |
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