第一部分：以碳酸酐異構酶II、IX及XII RNA干擾技術探討對人類乳癌細胞侵襲及移動能力的影響 第二部分：C型肝炎病毒套膜蛋白E2導致肝臟纖維化機制探討

Part I: The inhibition of migration and invasion in human breast cancer cells by carbonic anhydrase II, IX and XII RNA interference Part II: Molecular mechanism of hepatitis C virus E2-induced hepatic fibrosis

謝明儒(Ming-Ju Hsieh)

指導教授 ： 謝易修 ； 邱慧玲

中山醫學大學/醫學院/生化暨生物科技研究所/博士(2011年)

https://doi.org/10.6834/CSMU.2011.00115

摘要

第一部分
碳酸酐酶 （carbonic anhydrase；CA）是一種含鋅離子的金屬酵素，包含許多不同的異構酶，其功能主要是在於催化細胞內的酸性物質並維持細胞內的衡定。而其中存在於細胞質的CA II與細胞膜上的CA IX和CA XII則是被認為對於影響細胞內碳酸根離子的衡定以及維持細胞外pH值的正常最主要的影響因子之一。在先前的研究證實，在60個乳癌患者中，CA II的蛋白表現和CA活性在腫瘤部位有顯著的上升（P<0.001），而在mRNA層次也是和蛋白質表現有相同的結果。另外在探討乳癌組織中CA II活性與其臨床上一些數據的相關性分析也發現CA II活性和腫瘤大小有顯著的相關性並且也證實CA IX和CA XII在腫瘤發生的過程中是扮演著重要的角色。然而目前已經有許多的研究結果針對CA II以及CA IX對於癌細胞侵襲以及移動的能力進行探討，但是針對於CA XII的研究結果則尚未很清楚；根據先前的文獻中提到，CA XII的表現量在臨床手術預後情形的觀察追蹤上，是一個用來推測乳癌細胞侵襲的一個很好的指標。因此，在本研究過程中主要是先探討CA II、CA IX以及CA XII在不同的乳癌細胞中表現程度的差異性以及對於乳癌細胞侵襲以及移動能力的影響，並進一步的探討影響癌細胞侵襲以及移動能力的相關路徑。在研究中主要針對三株侵襲和移動能力程度不同的乳癌細胞進行研究，分別利用CA II、CA IX以及CA XII的RNA干擾技術來探討對於乳癌細胞侵襲以及移動能力的影響。結果發現，分別對MCF-7、Hs578T以及MDA-MB-231這三株乳癌細胞進行CA II、CA IX以及CA XII RNA干擾之後，以CA XII RNA干擾對於MDA-MB-231的侵襲以及移動能力的抑制最為明顯；進一步的利用轉殖成功後能持續穩定表現CA XII RNAi的MDA-MB-231細胞進行研究，結果發現會藉由抑制p38的磷酸化進而減弱MDA-MB-231細胞的侵襲以及移動能力；最後在活體動物的實驗中，將會穩定表現CA XII RNAi的MDA-MB-231細胞接種到動物體內觀察腫瘤大小以及重量。結果證實，在持續性的CA XII RNA干擾下，無論是腫瘤的大小以及重量都有明顯受到抑制的情形。根據這些實驗的結果證實，CA XII在MDA-MB-231乳癌細胞的侵襲以及移動能力上確實扮演著重要的角色。因此，在未來的治療腫瘤的方法上，對於CA XII有明顯表現的癌細胞當中，RNA干擾技術或許可以提供另一種新的應用方法。
第二部分
慢性肝炎及肝硬化一直是影響國人健康及造成死亡原因的主要原因之一。除了B型肝炎之外，C型肝炎也是主要引起慢性肝病的原因之一。C型肝炎是由C型肝炎病毒（hepatitis C virus；HCV）感染所引起的慢性肝臟疾病，與肝硬化及肝癌有著因果的關連性。文獻指出C型肝炎病毒套膜蛋白E2與肝臟星狀細胞膜上接受器CD81結合後，會藉由活化ERK/MAPK的途徑活化轉錄因子AP-2的活性，進而導致matrix metalloproteinase（MMP）-2的蛋白表現量以及活性上升，顯示E2蛋白與HCV導致的纖維化有關。另外，之前的文獻證實肝星狀細胞與肝纖維化的產生具有很重要的相關性。因此，本實驗主要探討C型肝炎病毒中的套膜蛋白E2對肝星狀細胞是否會造成相關纖維化因子表現的情形產生，並進一步的探討和纖維化之間的相關性以及影響的機制。本研究利用real-time PCR、西方墨點法、gelatin zymography、RNA干擾技術（small interfering RNA；siRNA）、流式細胞儀分析（flow cytometry）、免疫螢光染色（in situ immunofluorescence assay）和酵素聯結免疫吸附法（enzyme linked-immunosorbent assay；ELISA）等方法，證實套膜蛋白E2確實造成alpha smooth muscle actin (α-SMA)、collagen α（I）和connective tissue growth factor（CTGF）纖維化因子以及發炎因子interleukin（IL）-6、IL-1β表現量增加，也會促進transforming growth factor beta（TGF-β1）表現量增加，並刺激TGF-β1訊息傳遞路徑的啟動，而MMP-2表現量及活性的增加也更加證實套膜蛋白E2具有刺激肝星狀細胞活化的能力。最後進一步證實套膜蛋白E2會藉由刺激肝星狀細胞產生H2O2對細胞產生氧化性傷害，進而活化Janus kinase/signal transducers and activators of transcription (JAK/STAT)傳遞路徑、刺激ERK1/2和p38磷酸化而造成纖維化因子collagen α（I）表現量增加。研究結果證明套膜蛋白E2在C型肝炎病毒誘導纖維化產生的過程當中扮演了重要的角色，希望對於往後在C型肝炎病毒與纖維化的研究與治療上能提供新的研究方向。

關鍵字

碳酸酐酶乳癌 ； 細胞侵襲 ； 腫瘤轉移 ； 套膜蛋白E2 ； 纖維化 ； 氧化性傷害 ； 肝星狀細胞

並列摘要

Part: I
Carbonic anhydrase (CA) is an enzyme with zinc metal ions, including a number of different isoforms, and its main function lies within the catalytic acid cells and maintain the cell's internal balance. CA II, CA IX and CA XII are considered to affect the cell's internal balance of carbonate ions and maintain the normal extracellular pH. A previous study conducted with 60 breast cancer patients revealed a significant increase of CA II protein expression and CA activity in in tumor (P <0.001), as well as mRNA and protein expression levels. Furthermore, a significant correlation was found between CA II activity and tumor size while CA IX and CA XII are involved in the process of tumor development. Although there were extensive results indicating the involvement of CA II and CA IX in the cancer cell invasion and migration, similar studies for CA XII is less and insufficient. Previous studies have revealed that CA XII expression is an indicator for breast cancer cell invasion and useful for tracking surgical outcomes. Therefore, the aim of this study was to explore the impact of CA II, CA IX and CA XII on the extent of invasion and mobility in different breast cancer cells. Furthermore, the related pathways were alsoexplored. In the study with breast cancer cells of different levels of invasion and mobility, results from RNA interference technology showed that the CA XII RNA interference has the most significant inhibition capability for the invasion and migration of MDA-MB-231 cells. Further studies revealed that such inhibition is via an inhibition of phosphorylation of p38. In the final in vivo experiments, CA XII-knockdown MDA-MB-231 cells were inoculated into mice to show that the tumor size and animal body weight were significantly reduced in CA XII-knockdown mice. Take together, these results confirmed that CA XII indeed play an important role in the invasion and migration of MDA-MB-231 breast cancer cells.
Part: II
Chronic infection of hepatitis C virus (HCV) leads to hepatic fibrosis and subsequently cirrhosis, although the underlying mechanisms have not been established. Previous studies have indicated that the binding of HCV E2 protein and CD81 on the surface of hepatic stellate cells (HSCs) lead to the increased protein level and activity of matrix metallopeptidase (MMP) 2, indicating that E2 may involve in the HCV-induced fibrosis. This study was designed to investigate the involvement of HCV E2 protein in the hepatic fibrogenesis. Results showed that E2 protein may promote the expression levels of α-smooth muscle actin (α-SMA) and collagen α(I). Furthermore, several pro-fibrosis or pro-inflammatory cytokines, including transforming growth factor (TGF)-β1, connective tissue growth factor (CTGF), interleukin (IL)-6 and IL-1β, were significantly increased in E2 transfected-HSC cell lines, while the expression of MMP-2 are also considerably increased. Moreover, the significant increases of CTGF and TGF-β1 in a stable E2-expressing Huh7 cell line were also observed the same results. Further molecular studies indicated that the impact of E2 protein on collagen production related to higher production of ROS and activated Janus kinase (JAK)1, JAK2 and also enhance the activation of ERK1/2 and p38, while catalase and inhibitors specific for JAK, ERK1/2, and p38 abolish E2-enhanced expression of collagen α(I). Taken together, this study indicated that E2 protein involve in the pathogenesis of HCV-mediated fibrosis via an up-regulation of collagen α(I) and oxidative stress, which is JAK pathway related.

並列關鍵字

carbonic anhydrase ； invasion ； migration ； breast cancer ； hepatitis C virus E2 protein ； fibrosis ； oxidative stress ； collagen ； TGF-β ； JAK ； STAT

參考文獻

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