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系統識別號 U0026-2407201415242900
論文名稱(中文) Metformin處理之肝癌細胞中CEBPD誘發之細胞自噬及凋亡之探討
論文名稱(英文) Characterization of CEBPD-induced autophagy and apoptosis in metformin-treated hepatocellular carcinoma
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 102
學期 2
出版年 103
研究生(中文) 陳月秋
研究生(英文) Yueh-Chiu Chen
學號 S26014057
學位類別 碩士
語文別 英文
論文頁數 63頁
口試委員 指導教授-王育民
口試委員-張文昌
口試委員-張雋曦
口試委員-顏家瑞
中文關鍵字 CEBPD  LC3B  ATG3  細胞自噬  metformin  肝癌 
英文關鍵字 CEBPD  LC3B  ATG3  autophagy  metformin  HCC 
學科別分類
中文摘要 Metformin原本為第二型糖尿病人的用藥,近年來臨床調查發現病人服用metformin能降低其罹患肝癌的風險。進一步實驗結果指出,metformin能透過誘導肝癌細胞停滯在G1時期並進一步造成細胞凋亡,並且metformin能透過活化AMPK路徑活化細胞自噬的發生,然而目前對於metformin引發肝癌細胞凋亡及其與已知的細胞自噬角色關連仍未清楚。CCAAT/enhancer-binding protein delta (CEBPD)為一抑癌基因,本實驗室過去證實許多抗癌藥物能夠引發CEBPD基因表現量增加。本篇研究發現metformin可以穩定CEBPD的蛋白質穩定度而致其在細胞中的存在量增加。在缺乏CEBPD的情形下會減弱metformin抑制癌細胞生長的能力,並且降低細胞死亡的情形。重要的是,除了增加p27及降低cyclinD1的表現,CEBPD亦可活化參與細胞自噬作用中的LC3B和ATG3表現。上述結果指出CEBPD參與細胞自噬。進一步利用合併metformin和rapamycin處理肝癌細胞,可增強癌細胞的細胞凋亡情形。總結來說,CEBPD可透過調控p27、cyclinD1、LC3B和ATG3,參與在metformin所誘導的細胞生長停滯、細胞凋亡及細胞自噬作用中。並且我們也提供了一個藉由合併metformin和rapamycin的癌症治療方式,其在未來或許能夠成為臨床治療的方法之一。
英文摘要 Like many other cancers, the patients with hepatocellular carcinoma (HCC) face the risk of cancer relapse or liver dysfunction. Metformin, a first-line drug of type 2 diabetes, has been proved that it could induce G1 arrest and prevent or inhibit HCC. Moreover, metformin, an AMPK activator, could also activate cell autophagy. However, the connection between autophagy and metformin-mediated apoptosis is still unknown. Previous studies have demonstrated that the transcription factor CCAAT/enhancer-binding protein delta (CEBPD) plays a tumor suppressor and is response to many anticancer drugs in HCC. In this study, it found that metformin could induce autophagy, growth arrest and further apoptosis in Huh7 cells. Metformin enhanced CEBPD expression via stabilizing its protein stability. The loss of CEBPD attenuated the metformin-induced autophagy, growth arrest and apoptosis. Importantly, besides increasing p27 and down-regulating cyclinD1, CEBPD also contributed to the increases of LC3B and ATG3 gene expressions and LC3 puncta formation. These results suggested that the involvement of CEBPD in metformin-induced autophagy. Moreover, combined metformin with autophagy activator, rapamycin, elicits stronger cytotoxicity than single treatment alone in vitro. Taken together, we revealed a novel involvement of CEBPD in metformin-induced growth arrest, apoptosis and autophagy of liver cancer cells through regulating p27, cyclinD1, LC3B and ATG3. We also provide a new therapeutic approach by combination metformin with rapamycin on HCC.
論文目次 Contents
Abstract I
Abstract in Chinese II
Contents III
Figure Contents VI
Appendixes IX
Chapter 1 Introductions 1
1.1 Metformin 1
1.2 Autophagy 1
1.2.1 Autophagy 1
1.2.2 Cross-talk between autophagy and apoptosis 2
1.3 CCAAT/enhancer binding protein delta (CEBPD) 3
1.3.1 C/EBPs family 3
1.3.2 CEBPD as a tumor suppressor 3
1.4 Rapamycin 4
1.5 Combination therapy for cancer 5
1.6 Hepatocellur carcinoma (HCC) 5
1.7 Motivation 6
Chapter 2 Materials and Methods 7
2.1 Materials 7
2.2 Methods 8
Cell culture 8
Cell viability 8
Plasmid transfection and reporter assays 9
Reverse transcription polymerase chain reaction (RT-PCR) 10
Caspase-3/7 activity Assay 10
Lentiviral shRNA assay 10
Quantitative real time polymerase chain reaction (Q-PCR) 11
Fluorescence microscopy 12
Chromatin immunoprecipitation (ChIP) 12
Cell cycle analysis 13
Western blotting assay 13
mRNA half-life assay 14
Protein stability 14
Statistical analysis 14
Chapter 3 Results 15
CEBPD plays as a tumor suppressor in metformin-mediated anticancer effects in Huh7 cells 15
Metformin induces CEBPD via protein stabilization 16
Metformin induces autophagy in Huh7 cells 16
Autophagy is required for metformin-induced cell apoptosis in Huh7 cells 17
CEBPD is involved in metformin-induced autophagy 18
CEBPD binds to LC3B and ATG3 promoter and activates their expressions 19
Synergistic effects of metformin combined with rapamycin in vitro 21
Chapter 4 Discussions 22
References 26
Figures 35
Appendixes 62
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