- 學位論文
探討B型肝炎病毒之X蛋白促進肝癌轉移之機制:藉由調控上皮剪接調控蛋白1及2活化CD44v6的表現
Exploration of HBV X protein on the promotion of Hepatocellular carcinoma (HCC) metastasis: Through the activation of CD44 splice isoform switching by regulating splicing factor ESRP1/ESRP2
摘要
B型肝炎病毒(Hepatitis B virus, HBV)會造成肝癌發生的致病因素之一，且會促進肝癌細胞的移動及侵襲(Liver cancer cell migration and invasion)的發生。研究指出HBV X蛋白表現的肝癌細胞株可觀察到CD44家族成員的表現量提高，而CD44標準型(CD 44 standard, CD44s)存在於所有正常細胞，表現量提高會促進與細胞外基質的交互作用促進細胞骨架的重組及形態的改變而影響生長，而CD44變異型(CD44 variant, CD44v)則是與腫瘤的轉移有很大的關係。因此HBV X蛋白在肝癌移動及轉移的過程中是否可藉由改變癌細胞膜上CD44s及CD44v的表現量變化而促進肝癌轉移是本篇研究想探討的重點，此外也希望能了解在肝癌轉移的過程中HBV X蛋白是透過怎麼樣訊息傳遞機制去調控CD44s及CD44v的表現。本篇研究使用穩定表現HBV X蛋白的肝癌細胞株進行實驗，由西方墨點法觀察到HBV X蛋白會促進N-cadherin及抑制E-cadherin表現造成EMT現象的發生，而從幾丁質酶譜法(Gelatin zymography)可看到MMP2/9的酶切能力提高，並且也利用西方墨點法及流式細胞儀確定了HBV X蛋白會促進CD44及CD44v6的表現，由這些結果可觀察到HBV X蛋白會提高肝癌細胞的轉移能力。而從細胞電生理的癌細胞移動/侵襲實驗也確認了HBV X蛋白會提高肝癌移行/侵襲能力。我們的實驗結果也觀察到在HBV X蛋白調控肝癌轉移時RNA剪切過程(Splicing)的關鍵因子ESRP1和ESRP2的表現。更進一步發現，HBV X蛋白促進肝癌轉移是透過轉錄因子Snail而去抑制ESRP1和提高ESRP2的表現進而去促進CD44v6的產生而增加肝癌轉移的能力，或許未來能藉由調控ESRP1和ESRP2的表現來阻止B型肝炎病毒衍生的肝癌轉移的發生。
並列摘要
Hepatitis B virus (HBV) is one of the main aetiological agents for hepatocellular carcinoma (HCC), which elevates mortality rate due to its high incidence of metastasis. The genome X of HBV produces transcriptional trans-activating protein kinase, which is associated with hepatocarcinogenesis. Among cancer stem cell (CSC) markers, CD44 is associated with migration, proliferation, and invasion. In particular, CD44 variant 6 (CD44v6) is critical for metastasis. Therefore HBV x proteim (HBx protein) may contribute to the acquisition of metastatic properties of HCC by increasing their ability to bind hyaluronan acid (HA) through expressing different CD44 in the outer margin of the tumors. The current study aims to elucidate the role of HBx in regulating CD44v6 during HCC metastatic processes. We used HBx-transfected HCC cell lines to confirm the role of HBx in HCC invasion by enhancing epithelial-mesenchymal transition (EMT), matrix metalloproteinase (MMP) 2/9 activity, and CD44v6 expression by immunoblotting and flow cytometry. The metastatic ability of HBx-induced HCC cell lines was investigated using real-time cell analyzer to monitor migration/invasion ability. Our results confirmed that HBx protein is able to promote the metastatic ability in HCC cell lines. Also, we found that there was an regulated protein expression of epithelial splicing regulatory protein (ESRP) 1/2 on HBx-induced HCC cell lines. Furthermore, we also found that HBx regulates metastatic modification of CD44 variants and downstream signaling through the splicing factors -ESRP1 and ESRP2, which function as specific modulators in CD44 splicing, and in enhancing metastatic ability. Additionally, HBx alters the expression and function of ESRP1 and ESRP2, which may be a key element for regulating ECM interaction and degradation on CD44v6-induced metastasis. Regulation of ESRP1 and ESRP2 might be a potential approach to arrest the progression of HBV-related metastasis.
參考文獻
延伸閱讀
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