- 學位論文
探討組蛋白甲基轉移酶G9a在肝癌進程中的角色
Evaluation of the Role of Histone Methyltransferase G9a in Hepatocellular Carcinoma Progression
摘要
不正常的後遺傳調控 (epigenetic regulation) 時常導致疾病發生，包括癌症。癌細胞普遍呈現出不正常的DNA 甲基化 (methylation) 模式、組蛋白修飾 (histone modification)，以及非編碼RNA (non-coding RNA) 的表現。G9a 為哺乳動物的組蛋白甲基轉移酶，負責催化組蛋白H3 上第九個離胺酸 (K9) 的甲基化，組蛋白H3K9 的甲基化對腫瘤抑制基因轉錄的抑制扮演關鍵性的角色。已知在缺氧的情形下，會誘發G9a 表現及增加其甲基轉移酶活性進而調控基因表現。我們先前研究發現，在肺癌和卵巢癌的腫瘤組織，G9a 的表現程度高的患者其預後較差，並參與在轉移及侵襲過程中的分子調控。除此之外，過去研究指出，G9a mRNA 亦高度表現在肝癌病人的腫瘤組織中，但是目前對於其扮演的角色仍不清楚。因此，我們試圖探討G9a 在肝癌發展過程中的重要性及其調控機轉。在本研究論文中，我們發現相較於腫瘤周邊正常組織，G9a 高度表現在肝癌病人的腫瘤組織中，顯示其可能參與調控腫瘤的生成。利用特異性G9a 抑制劑BIX-01294 阻斷其組蛋白甲基轉移酶活性以及在G9a 高度表現之細胞株利用小片段干擾核醣核酸 (siRNA) 抑制G9a 表現，皆觀察到癌細胞存活率下降之情形，進一步探究原因發現，抑制G9a的癌細胞生長抑制與細胞自噬 (autophagy) 作用有關，同時偵測到ATG4 表現量上升，顯示G9a 可能藉由此分子途徑調控細胞自噬。動物實驗結果也證明，在持續表現siRNA 抑制 G9a 表現的肝癌細胞株可以顯著減緩腫瘤生長，並且可能透過誘發腫瘤的細胞自噬及抑制腫瘤血管新生來達成。另外，在對於多重激酶抑制劑sorafenib 具有抗性之肝癌細胞株中發現，G9a 表現量和生長速度均較親代細胞大幅增加，顯示出G9a 在癌細胞抗藥性發展過程中扮演重要角色。為了釐清G9a 所受調控情形，我們分析了可能調控G9a 的微型核糖核酸 (microRNA)，其中特異表現於肝臟之miR-122 可直接結合到G9a mRNA 的3’非轉譯區 (3’UTR) 並抑制G9a的表現。先前的證據顯示，慢性發炎和癌症進程之間的關連性，我們進而處理發炎反應相關的細胞激素IL-6 可誘發G9a 蛋白表現量增加，顯示G9a 的表現與發炎以及腫瘤微環境有所關聯。綜合以上研究，顯示G9a 在肝癌發展過程中扮演重要角色，未來有機會運用在抗藥性癌症的治療上。
並列摘要
Aberrations of epigenetic regulation influence many diseases involving in cancer.Cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in cancer including DNA methylation, histone modifications, nucleosome positioning and non-coding RNAs, specifically microRNA expression. G9a is a major mammalian H3K9 methyltransferase that contributes to the epigenetic silencing of tumor suppressor gene. It has been reported that hypoxia induced G9a protein expression and enzyme activity which leads to diverse transcriptional regulation of downstream functional genes. In our previous studies, elevated levels of G9a were correlated with poor prognosis in lung and ovarian cancer, and act as a regulator of genes involved in invasion and metastasis. In addition, it has been proposed that the mRNA expression of G9a in hepatocellular carcinoma (HCC) was higher than non-tumor tissue statistically, however, its functional and biological roles in HCC remains unclear. Hence, in this study we attempt to explore the functional roles of G9a and its possible regulatory mechanisms during HCC cancer progression. Here, we also found that G9a mRNA expression is up-regulated in HCC than non-cancerous liver tissue. These results indicate that G9a seems to be required for HCC carcinogenesis. Using specific G9a inhibitor – BIX-01294 or RNAi-mediated knockdown of G9a in HCC cells with higher endogenous G9a expression inhibited cell proliferation in vitro and tumor growth in vivo by induction of autophagy-associated cell death which is associated with up-regulation of ATG4. On the other hand, we observed that G9a protein up-regulated in sorafenib-resistant HCC cell line, knockdown of G9a in sorafenib-resistant HCC cell line abrogated this phenomenon. This finding indicates that high G9a protein level might lead to growth advantage of HCC cells under inhospitable condition and contribute to the development of drug-resistant cancer cells. To further understand the regulation of G9a up-regulation during cancer progression, we analyzed miRNA that can regulate the expression of G9a. miR-122, an liver-specific miRNA, can direct bind to 3’-UTR of G9a mRNA and thereby suppress G9a protein expression. As the evidence linking chronic inflammation to cancer progression, we found that treatment of inflammatory cytokine – IL-6 can induce the protein expression of G9a,indicating that regulation of G9a might correlate with inflammatory and tumor microenvironments. Together, G9a plays a crucial role during HCC cancer progression and might act as a therapeutic molecular target of drug-resistant cancer cells in the near future.
參考文獻
延伸閱讀
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