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系統識別號 U0026-0812200915271199
論文名稱(中文) 分析E2F1抑制CEBPD基因轉錄參與腫瘤形成之分子機轉與影響
論文名稱(英文) Dissection of the function of E2F1-induced CEBPD silencing in tumorigenesis
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 97
學期 2
出版年 98
研究生(中文) 吳宛臻
研究生(英文) Wan-chen Wu
電子信箱 s2696404@mail.ncku.edu.tw
學號 s2696404
學位類別 碩士
語文別 中文
論文頁數 52頁
口試委員 口試委員-黃阿梅
指導教授-張文昌
指導教授-王育民
口試委員-蔡坤志
中文關鍵字 tumorigenesis  PcG protein  E2F1  CEBPB  CEBPD 
英文關鍵字 CEBPD  CEBPB  hepatocarcinoma  E2F1 
學科別分類
中文摘要 文獻已證實,慢性發炎往往伴隨許多疾病(如:阿茲海默症、第二型糖尿病、癌症等)的生成。然而發炎反應導致癌症的生成及其機轉仍然尚未明瞭。CEBPD 為 CCAAT/Enhancer binding protein (C/EBP) 家族成員之一,與其家族成員CEBPB可結合至相同的C/EBP motif,並可共同調控下游基因的表現。近年的一些文獻報導以及實驗室先前的研究顯示 CEBPD 除了可以誘發細胞的生長停滯以及促進細胞的分化,並可受到促發炎激素刺激誘導大量的表現,也另外在肝脂生成扮演重要的角色。我們發現在一些子宮頸癌以及肝癌的病人組織中,CEBPD有靜默的情形,主要是因為SUZ12和EZH2高度表現,並促使DNMTs將CEBPD啟動子甲基化,進而導致CEBPD的靜默,這同時也意味著CEBPD靜默在癌症發展過程中可能扮演重要角色,另外,E2F1是個致癌因子的功能,並已被證實可以調控polycomb protein複合體中SUZ12、EZH2的基因轉錄,並在癌症發展中扮演著不可或缺的關鍵角色,因此, E2F1是否可藉由調控SUZ12及EZH2,導致CEBPD功能的喪失,最終促使細胞由發炎走向癌化,分子調控機制為何,是我們汲於想研究的目標。由實驗初步證實,當E2F1 過度表現於HepG2及Huh7細胞時,會活化下游的SUZ12及EZH2的基因轉錄,並專一地抑制CEBPD的轉錄表現,但不影響CEBPB的表現。同時,利用colony assay也發現,E2F1過度表現,可加強細胞轉型能力,但送入外源性的CEBPD,則可反轉這樣的現象,此外,藉由報告子分析實驗,發現在促發炎激素,可誘導CEBPD的表現,並可以促使下游與細胞凋亡、發炎相關基因的表現,有趣的是,在E2F1的過度表現下,則會抑制CEBPD啟動子的轉錄活性,推測此機制是細胞由發炎到癌化的關鍵因素。另外在利用報告子分析實驗也同時發現缺乏sumoylation的CEBPB可專一地促使COX-2啟動子的轉錄活性,由以上實驗結果,證實E2F1的過度表現,會抑制CEBPD的轉錄活性,在此同時,缺乏sumoylation修飾下的CEBPB會誘導COX-2的大量表現,增加細胞走向癌化的機會,進而導致癌症的發生。
英文摘要 Many studies reoprted that chronic inflammation is associated with diseases such as neuron degeneration, atherosclerosis, type 2 diabetes and cancer.However, an important issue for mechanism of the transient stage from inflammation to cancer remains less studied. CEBPD and CEBPB can recognize same C/EBP motif, and collaborate with each other to involve in the transcriptional regulations of the same genes. Up-regulation of CEBPD can respond to the pro-inflammatory-factors stimuli, and is involved in the regulation of hepatic lipogenesis. In addition, CEBPD is suggested to serve as a potential tumor suppressor. We found that CEBPD is down-regulated in cervical cancer and hepatocarcinoma cell through the epigenetic regulation by SUZ12/EZH2-mediated DNA/histone methylation. Therefore, these observations raised an interesting issue of whether the inactivation of CEBPD plays an essential role in the switch between inflammation and cancer. E2F1 is suggested to act as an oncogene, and was reported to activate the SUZ12/EZH2 transcription in cancers. However, the precise mechanism for the targets responding to the participation of E2F1 in the tumorigenesis, especially in the transient stage between inflammation and cancer, is less studied. Herein, we demonstrated that the E2F1 can specifically repress the CEBPD, but not CEBPB, transcription through the activation of SUZ12 and EZH2. Furthermore, the exogenously protein drove CEBPD, which is independently regulated by E2F1, could substantially inhibit the E2F1-induced colony formation. In reporter assay, loss-sumoylation of CEBPB can specifically promote COX-2 promoter transcription activity. These results suggested that the E2F1-mediated expression changes of CEBPD, and CEBPB combining the loss-of sumoylation effect can benefit the tumor.
論文目次 中文摘要 II
Abstract IV
致謝 VI
第一章 緒論 1
1. 發炎與癌症 1
2. CCAAT/enhancer-binding proteins (C/EBPs)之生理功能 2
3.E2F 轉錄因子家族的功能與特性 4
4.Polycomb group(PcG)蛋白質的功能和特性 4
5. Peroxisome proliferator-activated receptors γ,(PPARG)的功能與特性 6
6. GADD153的功能及特性 6
7. COX-2的功能與特性 7
8.研究動機 8
第二章 實驗材料 10
第三章實驗方法 14
1. 細胞培養 14
2. Oil red O 中性脂肪染色作用 14
3. 細胞全量RNA 萃取 15
4.反轉錄反應(reverse transcription, RT) 15
5. PCR 16
6.西方點墨法(Western blotting) 17
7.報導基因的分析(Reporter assay) 20
8.質體的建構 22
9. Foci formation assay 24
10. Colony assay 24
第四章 實驗結果 25
1.E2F1能專一性地抑制CEBPD基因的表現,並在癌化過程中扮演重要角色 25
2. CEBPD功能的喪失對於SUZ12和E2F1所誘導的細胞增生是不可或缺的條件 26
3.在高脂的刺激下,CEBPD功能的損失,會導致細胞有過度增生的情形 27
4.在發炎期間,CEBPD可以專一地誘導下游調控因子COX-2、PPARG2、GADD153啟動子的轉錄 28
5.在腫瘤生長過程中,缺乏sumoylation修飾的LAP1可專一地誘導發炎介質COX-2基因的轉錄 29
6. CEBPD為抑癌因子,並可廣泛地被抗癌藥物所活化 30
7.結論 31
第五章 討論 32
第六章 參考文獻 36
附圖 42
Fig. 1 E2F1 can play a specific role in repression of CEBPD gene 42
Fig. 2 Absence of CEBPD is important for SUZ12- and E2F1-induced cell proliferation 44
Fig. 3 Overexpression of CEBPD attenuates E2F1 induced cell proliferation and transformation. 46
Fig. 4 Loss of CEBPD can enhance HFD-induced cell proliferation 47
Fig. 5 Overexpressed E2F1 blocked CEBPD promoter activity in inflammatory state. 48
Fig. 6 CEBPD can specifically induce COX-2, PPARG2 GADD153 promoter activity in inflammatory states. 49
Fig .7 Sumo-deficient LAP1 can specifically induce COX-2 transcription in status of tumor formation. 50
Fig .8 Activation of CEBPD is a common target of anticancer drugs. 51
Fig .9 The hypothesis model of CEBPD transcription activity in inflammation and tumorigenesis.. 52
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