EB病毒蛋白質Zta與EBNA2調控Survivin表現之探究

Abstract

摘要 Survivin是屬於IAP (inhibitor of apoptosis protein) 蛋白質家族的一員，此類蛋白質家族可以藉著直接結合Caspase而抑制其活性，藉此抑制細胞凋亡 (apoptosis) 的進行。近年來諸多研究指出Survivin也可以幫助細胞週期 (cell cycle) 的進行與染色體的分裂。除此之外，Survivin已經被認為是非常重要的腫瘤標誌物 (tumor marker)，因其高度表現於快速增生的細胞，而不表現在分化完全的成人細胞，甚至幾乎所有的癌細胞都可以偵測到Survivin大量的表現。在先前實驗室研究中觀察到與EB病毒高度相關的鼻咽癌 (NPC) 和移植後淋巴增殖性疾病 (PTLD) 的組織切片，Survivin均有大量表現的現象，同樣在EB病毒感染B細胞後也能觀察到Survivin高度表現的現象。然而EB病毒究竟如何調控Survivin表現，則目前並不清楚，因此於本研究我們嚐試探索EB病毒調控Survivin的機轉。實驗中利用反轉錄聚合酶鏈反應 (RT-PCR) 與西方墨點法 (Western blotting)，我們發現EB病毒所表現的轉錄活化子，Zta和EBNA2可以增加Survivin mRNA與蛋白質的表現。除此之外，我們也證明Zta與EBNA2可以藉由活化Survivin啟動子增加Survivin的表現。而利用報導者分析法 (reporter assay)，我們發現Survivin啟動子上對於Zta調控Survivin啟動子重要的片段，並且在電泳位移分析法 (EMSA) 的實驗也證明Zta可結合Survivin啟動子上的ZRE (Zta response element)，這些結果指出Zta可能是透過結合至Survivin啟動子上的ZRE活化Survivin的啟動子。此外我們藉由細胞核質分離法 (Subcellular fractionation) 發現Zta所增加的Survivin主要表現在細胞核裡，目前已知當Survivin大量表現在細胞核裡可以幫助細胞週期的進行與細胞的增生。而在[3H]胸腺嘧啶嵌入法實驗中我們也發現，表現Zta的細胞其生長速率較控制組細胞迅速。但是當我們利用siRNA的技術抑制Survivin的表現後，即使細胞表現Zta也無法幫助細胞增生，此結果顯示Zta可藉由增加Survivin表現促進細胞的增生。

Abstract Survivin is a member of IAP (inhibitor of apoptosis protein) protein family, which can inhibit apoptosis by directly binding and blocking activity of caspases. Recently, several studies reported that Survivn can promote cell cycle progression and sister chromatids segregation. Additionally, Survivin has been identified as a potential tumor marker, since it selectively overexpressed in most of cancer cells but not in normal cells. Previously, we found that Survivin was overespressed in nasopharygenal carcinoma (NPC) and in post-transplanted lymphoproliferative disease (PTLD) biopsies, and that overexpression of Survivin is highly associated with Epstein-Barr virus (EBV) infection. However, the mechanism of how EBV regulates Survivin expression is still unclear. Thus, we sought to investigate the mechanism of EBV regulates expression of Survivin. To aim this, several EBV viral proteins, including latent and lytic proteins, were transfected into 293 cells, and subjected to RT-PCR and western blotting to investigate their effects on the expression of Survivin. Two EBV viral transactivators, Zta and EBNA2 were found to enhance Survivin expresision in mRNA and protein levels. In addition, Zta and EBNA2 can activate Survivin promoter in luciferase reporter assay. Furthermore, we defined the crucial domain for Zta-mediated Suvivin promoter activity by luciferase reporter assay and demonstrated that Zta can directly bind to the Zta responsive element (ZRE) embeded in Survivin promoter by EMSA. These results suggest that Zta may upregulate Survivin expression through directly enhancing Survivin promoter activity. Of note, we found that Zta can specifically upregulate Survivin in the nuclear portion by subcellular fractionation assay, which is the sign for Survivin to promote G1/S phase progression and cell proliferation. Accordingly, we found that Zta can enhance cell proliferation rate in [3H]-thymidine incorporation assay. And, when we knocked down Survivin expression by siRNA (small interference RNA), This Zta-induced cell proliferation could be disrupted by Survivin siRNA. The results indicate that Zta can enhance cell proliferation via increasing Survivin expression.