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研究生:陳俊名
研究生(外文):Chun-Ming Chen
論文名稱:探討轉移性肝癌中hsa-miR-122之分子調控機制
論文名稱(外文):Molecular regulation of hsa-miR-122 in metastatic
指導教授:鄒安平
指導教授(外文):Ann-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:2007
畢業學年度:95
語文別:中文
論文頁數:66
中文關鍵詞:轉移肝癌微核糖核酸-122
外文關鍵詞:metastasishepatocellular carcinomamiR-122
相關次數:
  • 被引用被引用:1
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微核糖核酸(miRNAs)廣泛地涉及在人類的疾病及生物功能的層面。利用高產能的分子技術平台快速地產生出各種人類癌症組織中的miRNA表現圖譜及基因體圖譜後,可以用來鑑定與癌症有關的miRNA(oncomir)。雖然miRNA的表現最初會受到轉錄層次的控制,但是近來的證據也顯示,許多oncomir的基因其實是位在癌症基因體中異常的基因座上,而且在人類癌症中,這些oncomir的表現與染色體的增幅或缺失有直接的關連性。因為miRNAs會抑制他們目標基因的表現,故鑑定出oncomir的目標基因,便成為瞭解癌症形成機制的關鍵之一。
人類的hsa-miR-122是特異性存在於肝臟中的miRNA。在之前的研究中,我們發現發生肝內轉移的肝癌樣本中的hsa-miR-122表現量下降;當大量表現miR-122至肝癌細胞株中,則會降低細胞的移動能力,這暗示著hsa-miR-122在轉移性肝癌中扮演重要的角色。雖然有其他人的研究顯示,miR-122可能牽涉調控多種肝臟代謝的途徑,但hsa-miR-122在肝臟生理功能中的角色仍未清楚。故此篇研究之主旨即在討論miR-122在人類肝癌中的分子調控機制。
根據人類染色體18q21.31上的基因體資訊,我構築了兩種能夠透過核醣核酸聚合酶Ⅱ(RNA Pol.Ⅱ)轉錄出hsa-miR-122的表現載體。Hsa-miR-122在細胞移動及細胞侵襲能力的角色,已經透過細胞轉染或VSV-pseudo-lentivirus感染細胞的方式,大量原位表現在兩株肝癌細胞-SK-HEP-1及Mahlavu中得到驗證。大量表現miR-122除了降低了特定肝癌細胞株的移動、侵襲能力,hsa-miR-122對於細胞增殖及形態特徵的變化,則利用lentivirus系統做了較長時間的探討。 SK-HEP-1和Mahlavu細胞在受到miR-122-lentivirus感染之後的第五天,細胞形態明顯變得扁平且不再細長,而這些形態變化似乎和細胞移動能力成正比;大量表現miR-122至HuH7九天後,可以看到細胞週期停滯在G0/G1期的生長抑制情形。而我們也將探討上皮-間質轉化(EMT)的特徵變化。
我們預測出四十六個miR-122的下游基因,其中的十四個基因已進行過報導基因實驗。回復miR-122的表現可能足以抑制體內肝癌的轉移,故利用miR-122-lentivirus 的系統對於產生穩定表現miR-122的肝癌細胞供活體研究使用而言,將有相當大的裨益。
MicroRNAs (miRNAs) are found to participate in diverse aspects of biological functions and human diseases. High-throughput molecular genomic techniques rapidly generated expression and genomic profiles of miRNA in various human cancer samples and led to the identification of oncomir, miRNA with a significant biological role in cancer. Although expression of miRNA is primarily controlled at the transcriptional level but current evidence also revealed that many oncomir genes are located at aberrant loci of cancer chromosomes, with which oncomir expression is linked to chromosomal deletion or amplification in human cancers. Since miRNAs negatively regulate their targets, identification of oncomir targets becomes a key to understanding of carcinogenesis.
Hsa-miR-122 is a liver specific miRNA. Previously, we identified the down-regulation of miR-122 in intrahepatic metastastic HCC and overexpression of miR-122 in HCC cell lines reduced cell migration activity in vitro, suggesting a critical role of miR-122 of liver tumors in metastasis. Although hsa-miR-122 was shown to be involved in regulation of multiple liver metabolic pathways by others, the physiological roles of hsa-miR-122 in liver are not well understood. In this report, molecular regulation of hsa-miR-122 in human hepatocellular carcinoma (HCC) is examined.
Based on the genomic information of 18q21.31 (UCSC Genome Browser), two expressing vectors carrying hsa-miR-122 with RNA polymerase II promoters were constructed. A role of hsa-miR-122 in cell migration was confirmed by ectopically overexpressing miR-122 either with transfection of the expression plasmid or infection of VSV-G-pseudo-lentivirus in two HCC cell lines, SK-HEP-1 and Mahlavu. Overexpressing miR-122 reduced migration of selected HCC cells, and the effects of hsa-miR-122 on cell proliferation and morphological features were also studied with lentiviral system. SK-HEP-1 and Mahlavu became flattened five days after miR-122-lentivirus infection. The morphological changes seem to have a positive correlation with reduction of cell migration activity. Cellular growth rate of HuH7 was inhibited nine days after miR-122-lentivirus infection, and a block of cell-cycle at Go/G1 phase was observed. Alteration of EMT features was investigated.
Forty-six putative targets of miR-122 were identified, of which more than 14 genes were experimentally validated. It is possible that restoration of miR-122 expression may inhibit in vivo liver tumor metastasis. The lentiviral-122 system will facilitate in vivo studies with HCC cells stably expressing miR-122.
中英文名詞縮寫對照表 ………………………………………………iii
中文摘要 ………………………………………………………………iv
英文摘要 ………………………………………………………………vi緒論 ……………………………………………………………………1
論文研究動機 ………………………………………………………6
研究目的………………………………………………………………7
實驗材料 ……………………………………………………………8
實驗方法 ……………………………………………………………13
實驗結果 ……………………………………………………………24
討論 …………………………………………………………………31
結論與未來展望 ……………………………………………………38
參考文獻 ……………………………………………………………39
圖表 …………………………………………………………………43
圖一、定義hsa-miR-122之基因………………………………………43
圖二、MiR-122之表現載體及表現miR-122後對目標基因的影響…44
圖三、MiR-122及NUMBL調控細胞移動的能力………………………45
圖四、驗證miR-122對目標基因的調控………………………………46
圖五、以lentivirus系統大量表現miR-122於肝癌細胞中…………47
圖六、Lenti-122對於細胞形態之影響………………………………48
圖七、Lenti-122對細胞生長之影響…………………………………50
圖八、Lenti-122對細胞移動與侵襲能力之影響..…………………51
圖九、Lenti-122對HuH7細胞週期的影響……………………………52
圖十、Lenti-122對腫瘤形成能力的影響……………………………53
表一、預測miR-122之目標基因的數值變量及相關資訊。…………54
附錄 …………………………………………………………………55
附圖一、篩選miR-122的目標基因之策略及流程……………………55
附圖二、MiR-122在肝癌病人、小鼠肝臟及細胞株中的表現量……56
附表一、MiR-122目標基因的分子功能列表…………………………57
附表二、設計引子所用的序…………………………………………58
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