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研究生:江謝佩妘
研究生(外文):Pei-Yun Chiang Heish
論文名稱:探討HCC中hsa-miR-122轉錄調控機制
論文名稱(外文):Transcriptional regulation of hsa-miR-122 in HCC
指導教授:鄒安平鄒安平
指導教授(外文):Ann-Ping TsouAnn-Ping Tsou
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:72
中文關鍵詞:轉錄調控肝癌微核醣核酸
外文關鍵詞:miR-122HCCmiRNAtranscription
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微核醣核酸(microRNAs)具有多樣化生物功能,能抑制基因表現,尤其在生物發育、分化及癌症形成中扮演一個重要的調控者。在之前實驗室研究發現肝內特異性miRNA,hsa-miR-122,在正常肝臟中大量表現,但在肝內轉移之樣本中則表現非常微弱,且其表現量與肝癌惡化的程度成反比。此外,當大量表現miR-122時會抑制腫瘤的形成。此研究結果證實miR-122在肝臟組織中為一具功能性的腫瘤抑制miRNA。因此在本篇論文中,我將探討miR-122在肝癌中的轉錄調控機制。

我們首先定義出pri-miR-122的轉錄單位,包含其轉錄起始點和核心啟動子區域。在核心啟動子區域(+1~-180)中包含兩個TATA序列、兩個CCAAT序列以及一群FOX家族的轉錄因子結合位。當中利用shRNA沉寂FOXQ1基因表現,發現會些微影響miR-122基因表現,推測FOXQ1在pri-miR-122表現中為一可能調控者。由於在miR-122啟動子及intron區域(-309~+3054)沒有CpG島的序列,但擁有許多CpG二核苷酸序列。在HCC細胞株中,利用bisulfite處理,發現這些CpGs的甲基化程度與miR-122的基因表量成正比。在內生性會表現miR-122的HuH7細胞株中,這些CpGs完全去甲基化。當以去甲基化藥物(5-azacytidine)處理HepG2細胞株96小時後可誘發miR-122表現。且此基因活性的轉變可在此段區域之CpGs甲基化情形中,幾乎完全從甲基化轉變成去甲基化。在HCC細胞株中唯有HuH7和Hep-3B表現少量miR-122,而此差異推測是由於細胞株中存在不同的轉錄因子及染色體結構不同所造成。對於是否有染色體結構上的差異正在測試中。利用去甲基化藥物活化腫瘤抑制基因,miR-122,來達到負調控目標腫瘤基因,可作為一個新穎策略,運用於預防及治療人類肝癌的發生。
MicroRNAs (miRNAs) are important regulators of gene expression in multiple biological processes including development, differentiation and carcinogenesis. Previously we demonstrated that expression of liver-specific hsa-miR122 (miR-122) is abundant in the normal livers but is significantly down-regulated in liver cancers with intrahepatic metastasis. In addition, overexpression of miR-122 suppresses tumor formation. These results indicate that miR-122 is a functional tumor suppressor in liver tissue. The aim of this study is to understand how pri-miR122 is transcriptionally regulated.
We initially identified the complete transcription unit of pri-miR122 including its transcriptional start site and a functional core promoter region which drives specific expression of miR-122 in HCC cell line, HuH7. The core promoter region (+1 ~ -180bp) contains two TATA boxes, two CCAAT boxes and the binding sites for several potential transcription factors. Knockdown of FOXQ1 seems to result in less expression of miR-122, suggesting a possible role of FOXQ1 in regulating pri-miR122 expression. While no canonical CpG islands identified, we nonetheless located multiple CpG dinucleotides in the upstream and the intron regions (-309 ~ +3054bp) of pri-miR-122. The methylation status of these CpGs correlated with the miR-122 expression in selected HCC cells. In miR-122-expressing HuH7 cells, those CpGs are completely unmethylated. After 96-h treatment of 5-azacytidine, HepG2 cells started to express miR-122 among several HCC lines tested. The altered gene activity in HepG2 cells is accompanied with a near-complete conversion of the methylated CpGs within that region. The differential activation of miR-122 in different HCC cell lines is likely due to differential availability of transcription factors and possibly the chromatin structure. The effect of other chromatin-modifying drugs is under investigation. Activation of tumor suppressor miR-122 by demethylation drugs may cause downregulation of target oncogenes and could be a novel strategy for the prevention and treatment of human liver cancer.
中英文名詞縮寫對照表.……………………………………………… 1
中文摘要 .…………………………………………………………… 2
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緒論……………………………………………………………………. 4
論文研究動機………………………………………………………… 10
研究目的 …………………………………………………………… 11
實驗材料 …………………………………………………………… 12
實驗方法 …………………………………………………………… 17
實驗結果 …………………………………………………………… 30
討論 ………………………………………………………………… 37
總結與未來展望 …………………………………………………… 41
參考文獻……………………………………………………………… 42
圖表…………………………………………………………………… 49
附錄…………………………………………………………………… 68
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林晴雯(2006) 探討肝癌中的oncomiRs:miR-122於肝癌之調控,國立陽明大學醫學生物技術研究所碩士論文

李曜珊(2006) 探討轉錄因子FOXQ1在轉移性肝癌中所扮演的角色,國立陽明大學醫學生物技術研究所碩士論文

陳俊名(2007) 探討轉移性肝癌中has-miR-122之分子調控機制,國立陽明大學醫學生物技術研究所碩士論文

蔡偉智(2008) 探討轉移性肝癌中hsa-miR-122之分子調控機制,國立陽明大學醫學生物技術研究所碩士論文
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