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標題: | 腫瘤轉移抗原1在 B 型肝炎病毒相關肝細胞癌的角色 Roles of Metastatic Tumor Antigen 1 in Hepatitis B Virus-related Hepatocellular Carcinoma |
作者: | Yung-Tsung Li 李泳璁 |
指導教授: | 劉俊人 |
共同指導教授: | 吳慧琳 |
關鍵字: | 肝細胞癌,腫瘤轉移抗原1,B型肝炎病毒,土撥鼠,剪接異構體,RNA穩定度, hepatocellular carcinoma,metastatic tumor antigen 1,hepatitis B virus,woodchuck,splicing variant,RNA stability, |
出版年 : | 2017 |
學位: | 博士 |
摘要: | 肝癌 (hepatocellular carcinoma, HCC) 是一高致死率的癌症,主因是發現早期肝癌及有效治療晚期肝癌的方法相當有限。由於肝癌發展的分子機制具有高度異質性,再加上肝癌病人的臨床結果都非常不同,因此,有必要對不同分子特性的肝癌做深入研究,以發展新的診斷治療標記及最適性的個人化醫療。腫瘤轉移抗原1(MTA1)是一個在許多生理及病理現象中都扮演重要角色的分子,它在 B 型肝炎病毒相關肝癌 (HBV-HCC) 過度表現比例遠高其它病原 (etiology) 造成的肝癌,且與肝癌的侵略性及預後不佳高度相關,具有預測肝癌臨床表現及作為診斷治療標的的潛力,但 MTA1 為何在HBV-HCC特別容易過度表現及MTA1參與肝癌發展過程的分子機制還不清楚。在此論文中,我們利用人類肝細胞株、人類臨床檢體及 B 型肝炎相關肝癌的土撥鼠動物模式來探討MTA1在HBV-HCC的分子機制及可能的臨床意義。 第一部分,我們探討土撥鼠動物模式是否適用於研究MTA1在HBV-HCC過度表現及與肝炎病毒X蛋白交互作用的角色。我們透過選殖土撥鼠MTA1 (wk-MTA1) cDNA並且研究其分子功能,以提供我們研究MTA1在土撥鼠動物模式中HBV-HCC作用。wk-MTA1蛋白的序列和組成在不同物種間具高度保留性。在土撥鼠HCC中,wk-MTA1的 mRNA和蛋白質都是過度表現,這個結果與MTA1 在人類HCC中的表達相似。此外,MTA1剪接異構體,wk-MTA1dE4,在土撥鼠HCC中也是過度表達,而且它的表現量約佔總轉錄體(total transcripts)的50%。我們若針對wk-MTA1的表現量進一步分析,結果發現wk-MTA1dE4過度表達的土撥鼠HCC的百分比高於total wk-MTA1過度表達的土撥鼠HCC的百分比(77.8% vs. 61.1%),因此,這個結果顯示在土撥鼠HCC中wk-MTA1dE4可能是比 total wk-MTA1 更敏感的指標。在細胞株的研究中,我們在土撥鼠HCC細胞株(WCH17)中過度表達或敲低 (knockdown) wk-MTA1的表現,結果發現 wk-MTA1可以與土撥鼠肝炎病毒 X 蛋白 (WHx) 相互作用,並且證實wk-MTA1 蛋白在WHx介導的NF-κB活化和增進肝癌細胞移動及侵襲的能力中具有關鍵的作用。 第二部分,我們探討MTA1的相互作用蛋白-類胰島素生長因子II mRNA結合蛋白1(insulin-like growth factor II mRNA-binding protein 1, IGF2BP1)與MTA1 的相互作用在細胞中的角色。IGF2BP1是一種 RNA結合蛋白(RNA binding protein),它被證實會影響致癌基因的mRNA穩定性,並且可以增強腫瘤細胞的遷移能力。我們證實MTA1可以直接與IGF2BP1進行交互作用,而且是RNA-independent的方式。透過 in vivo RNA-immunoprecipitation (RIP),我們發現MTA1和IGF2BP1一樣,都能和IGF2BP1已知的標的基因的mRNA結合在一起。此外,Sequential RIP的實驗顯示MTA1可以與IGF2BP1和MYC mRNA形成ribonucleoprotein (RNP) complex。我們透過建立MTA1 敲低的(MTA1-knockdowned)SK-HEP-1肝癌細胞株,發現當MTA1表現降低時,MYC 無論在mRNA或蛋白質的表現水平上都會降低。我們透過mRNA decay assay,進一步探討MTA1是否會影響 MYC mRNA 的穩定度,我們發現MTA1-knockdowned SK-HEP-1細胞中MYC mRNA的穩定度會下降,因此,MTA1在維持MYC mRNA的穩定度上扮演角色。藉由 in vitro RNA-pull down,我們確認了MTA1具有直接結合RNA的能力。在人類肝癌組織中,MTA1與MYC的mRNA表現量之間呈現正相關。 第三部分,我們利用回溯性的資料分析,探討MTA1在HBV-HCC患者手術治療後的腫瘤復發的臨床意義。此研究共納入了102例接受肝腫瘤切除術的HBV-HCC患者。我們發現人類MTA1dE4過度表達HCC的百分比高於總MTA1過度表達的HCC的百分比(49% vs. 20.6%),這個結果與土撥鼠HCC的發現相似。在平均追蹤的48.2個月中,有67 位(65.7%)HBV-HCC患者腫瘤復發,其中有25位是早期腫瘤復發(early recurrence; 復發時間小於等於一年)和42位是晚期腫瘤復發(late recurrence; 復發時間大於一年)。此外,我們發現MTA1dE4過度表達的HBV-HCC患者具有較高的腫瘤復發率(tumor recurrence rate),尤其是早期復發。根據Stepwise multiple COX proportional hazards regression analysis的分析結果,當HBV-HCC患者具有腫瘤直徑大於3 公分[風險比(Hazards ratio, HR):10.165; 95% 信賴區間 (confidence interval, CI): 1.366-5.665; P = 0.024] 和血清中甲型胎兒蛋白(alpha fetal protein, AFP)水平 ≥ 200ng/ mL [HR: 4.028; 95% CI: 1.618-10.03; P = 0.03] 有較高的早期腫瘤復發的風險,顯示腫瘤直徑大於3公分和高血清甲型胎兒蛋白是早期復發的獨立預測因子。透過分層分析(stratified analysis),把肝硬化(liver cirrhosis)的效應列入考慮後,在肝硬化患者中腫瘤直徑大於3公分和早期復發風險之間有強烈關聯 [HR:10.629; 95% CI: 1.425-79.31; P = 0.021]。然而,高血清甲型胎兒蛋白和HBV-HCC中過度表達MTA1dE4則是呈現邊緣性顯著(borderline significance) (P = 0.059和P = 0.083)。 綜合上述三部分的研究,我們的研究結果支持了土撥鼠HCC是適合探討MTA1在HBV-HCC分子特徵的動物模式。此外,我們的研究探討MTA1在 HBV-HCC的分子機制和臨床意義,為 MTA1及其剪接異構體 MTA1dE4 的重要性提供了線索。我們首次揭露MTA1以前從未被報導過的結合RNA的新功能,並且發現MTA1在細胞質的潛在功能及穩定致癌基因mRNA的新致癌機制。因此,本論文的研究成果我們認為有助於未來闡明MTA1在HBV-HCC的分子機制和臨床意義,及優化MTA1過度表達的HBV-HCC的治療策略並發展最適性的個人化醫療奠定重要基礎。 Lack of sensitive biomarkers for timely diagnosis and the absence of effective therapeutics for treatment are the two major reasons for poor outcome in patients with advanced hepatocellular carcinoma (HCC). The molecular mechanism underlying HCC is highly heterogeneous and the clinical outcome of HCC is very variable among patients. Hence, it is necessary to study the molecular mechanisms for different etiologies of HCC to develop specific new markers for diagnosis and therapy. Metastatic tumor antigen 1 (MTA1) is a multifaceted master coregulator that plays critical roles in the physiological and pathological processes. MTA1 overexpression is more frequent in HBV-related HCC (HBV-HCC) than in HCC of other etiologies. Furthermore, it is highly associated with the invasiveness and poor prognosis of HCC. However, the underlying mechanisms for the higher percentage of MTA1 overexpression in HBV-HCC and the roles of MTA1 in HBV-associated hepatocarcinogenesis remain elusive. The aim of this dissertation is to investigate the molecular functions and clinical implications of MTA1 in HBV-HCC by use of human liver cell lines, clinical samples and woodchuck HCC model. In the first part of this study, we examined the appropriateness of using the woodchuck model for studying the roles of MTA1 in HBV-HCC with respect to MTA1 overexpression and molecular interaction with the hepadnavirus X protein. To investigate the roles of MTA1 in HBV-associated hepatocarcinogenesis in the woodchuck model, we cloned the woodchuck MTA1 (wk-MTA1) complementary (c)DNA and characterized its molecular functions. The sequence and organization of the wk-MTA1 protein were highly conserved among different species. Similar to its expression in human HCC, wk-MTA1 was upregulated in woodchuck HCC, as determined at RNA and protein levels. Furthermore, an MTA1-spliced variant, wk-MTA1dE4, was overexpressed in woodchuck HCC, and it was attributed to approximately 50% of the total transcripts. The percentage of wk-MTA1dE4-overexpressed woodchuck HCCs was higher than that of the total wk-MTA1-overexpressed HCCs (77.8% vs. 61.1%) and wk-MTA1dE4 may represent a more sensitive marker than the total wk-MTA1 in woodchuck HCC. We overexpressed or knocked down wk-MTA1 in a woodchuck HCC cell line and demonstrated that wk-MTA1 could interact with the WHV X protein (WHx) and play indispensable roles in WHx-mediated NF-κB activation and tumor cell migration- and invasion-promoting activities. In the second part of this study, we examined the roles of MTA1-interacting protein, IGF2BP1 (insulin-like growth factor II mRNA-binding protein 1) in MTA1-mediated functions in vitro. IGF2BP1 is an oncofetal RNA-binding protein (RBP) and has been shown to influence the mRNA stability of several oncogenes and can enhance the migration ability of tumor cells. We found that MTA1 could directly interact with IGF2BP1 in an RNA-independent manner. By in vivo RNA-immunoprecipitation (RIP) assay, we found that MTA1 could associate with the known IGF2BP1 target mRNAs. In addition, using sequential RIP, we demonstrated that MTA1 could form a ribonucleoprotein complex with IGF2BP1 and MYC mRNA. Using knockdown experiments, we showed that both mRNA and protein expression levels of MYC in MTA1-konckdowned SK-HEP-1 cells were decreased. Furthermore, by use of mRNA decay assay, we found that the half-life of MYC mRNA in MTA1-konckdowned SK-HEP-1 cells was decreased. By in vitro RNA-pull down, we confirmed that MTA1 exhibites direct RNA-binding ability. A positive correlation between MTA1 and MYC mRNA expression levels in human clinical HCC samples could be illustrated. These results suggested that MTA1 plays an integral role in stabilizing mRNA of MYC. This study reveals the novel RNA-binding function of MTA1 and provides important information to the molecular mechanism of MTA1 in cytoplasm and oncogenesis. In the third part of this study, we characterized the clinical significance of MTA1 overexpression in postoperative recurrence of HBV-HCC. In this retrospective cohort study, a total of 102 patients with HBV-HCCs underwent hepatic resection were enrolled. We found that the percentage of human MTA1dE4-overexpressed HCCs was higher than that of the total MTA1-overexpressed HCCs (49% vs. 20.6%), which was similar to those findings in woodchuck HCCs. During a mean follow-up period of 48.2 months, patients with MTA1dE4-overexpressed HCCs had a significantly higher rate of tumor recurrence, especially in early recurrence. Stepwise multiple COX proportional hazards regression analysis showed that large tumor size (over 3-cm in diameter) [hazard ratio (HR), 10.165; 95% confidence interval (CI), 1.366-5.665; P= 0.024] and high serum AFP level (≧ 200 ng/mL) [HR, 4.028; 95% CI, 1.618-10.03; P= 0.03] were associated with early tumor recurrence in HBV-HCC patients. Stratified analysis showed that larger tumor size was still associated with early recurrence in cirrhotic patients (HR, 10.629; 95% CI, 1.425-79.31; P= 0.021), whereas high serum AFP level and high MTA1dE4 expression were associated with borderline significance (P= 0.059 and 0.083). In summary, our results support the hypothesis that woodchuck HCC recapitulates HBV-HCC with respect to the molecular characteristics of MTA1 and provides new clues for conducting mechanistic studies of MTA1 in HBV-associated hepatocarcinogenesis, including the possible clinical significance of MTA1dE4. Our study also reveals the novel RNA-binding function of MTA1 and potentially provides a paradigm for a broader understanding of the molecular function of MTA1 in the cytoplasm and oncogenic activities. Therefore, results of this dissertation lay the groundwork for elucidating the detailed molecular mechanisms and clinical implications of MTA1 and optimizing therapeutic strategy to treat MTA1-overexpressed HBV-HCC in the era of personalized medicine. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67492 |
DOI: | 10.6342/NTU201702230 |
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顯示於系所單位: | 臨床醫學研究所 |
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