EB病毒基因表現與病毒致癌蛋白LMP2A在調控人類尿嘧啶雙磷酸葡萄糖去氫酵素基因及細胞移行的功能

Epstein-Barr Virus Gene Expressions and the Role of the Viral Oncoprotein LMP2A in Regulation of Human UDP-Glucose Dehydrogenase Gene and Cell Migration

潘韻如(Yun-Ru Pan)

指導教授 ： 張晃猷

國立清華大學/生命科學院/生命科學系/博士(2008年)

https://doi.org/10.6843/NTHU.2008.00533

摘要

Epstein-Barr virus (EBV) has been implicated in the development of many human neoplasias including B lymphoma and nasopharygeal carcinoma (NPC). During infection, EBV has the potential to undergo latent and lytic pathways. However, expression of many of the viral genes during the lytic-latent transition remains unclear. In the first part of my study, I investigated the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and hydroxyurea (HU), two commonly used EBV life cycle modulators, on the expression profiles of the entire genome of EBV persistent infected in B95-8 cells. After treatment with TPA for 48 h, the copy number of EBV genome in the cells increased about 2.5 fold, whereas HU treatment resulted in a reduction to approximately two-thirds of the original level. Except a small set of genes, the amounts of EBV mRNA are generally less abundant than that of □-actin. The expression of a large fraction of the 80 EBV genes was found to be activated after TPA treatment; in contrast, treatment of the B95-8 cells with HU, a nucleotide synthesis inhibitor, dramatically suppressed the expression of EBV lytic genes. Overall, this study demonstrated that quantitative real-time PCR is a reliable method to monitor the influence of drug-treatment in EBV genes regulation.
The EBV latent membrane protein 2A (LMP2A) is frequently detected in NPC biopsies. A recent cDNA microarray assay notes that expression of the UDP-glucose dehydrogenase (UGDH) gene shows high correlation with LMP2A levels in NPC biopsies. UGDH catalyzes oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor of extracellular matrix (ECM) component. Overproduction of ECM is associated with many cellular behaviors including motility and migration, suggesting that UGDH is a good target for controlling cancer metastasis. In the second part of my study, I had extended the findings and demonstrated that the UGDH transcript and protein quantities, the enzyme activity, and glycosaminoglycan contents increase in LMP2A over-expressed HEK293 cells. Results showed that a region from 630 to 486 bp upstream of the transcription start of UGDH promoter is critical for LMP2A mediated gene expression and an Sp1 binding site mutation in this region reduces the LMP2A-responsive expression of the UGDH gene. Moreover, cell motility enhancement by LMP2A diminishes by treating the cells with Sp1-specific inhibitor and siRNA. Using a signaling pathway specific inhibitor, it is revealed that PI3K/Akt and ERK, not JNK and p38, participate in LMP2A-induced UGDH expression. This study provides a molecular model of the mechanism participating in LMP2A-mediated UGDH gene activation.
In the third part study employed small interference RNA (siRNA) to downregulate human UGDH gene expression in colorectal carcinoma HCT-8 cells. Reduction in both mRNA and protein levels of UGDH could be clearly observed in siRNA-transfected HCT-8 cells. Production of glucosaminoglycans was also decreased in monolayer cells and spheroid culture upon transfection with the UGDH siRNA. The use of UGDH siRNA significantly disrupts HCT-8 cell invasive ability in both a three-dimensional collagen gel assay and transwell assay suggesting a possibility of UGDH to be used as a target for colorectal cancer therapeutic intervention.

關鍵字

EB病毒 ； 尿嘧啶雙磷酸葡萄糖去氫酵素 ； 潛伏第二A型膜蛋白 ； 基因表現 ； 細胞移行

並列摘要

EB病毒感染已被證實和許多人類細胞癌化有關，其中包括了B細胞淋巴癌和鼻咽癌。在EB病毒感染過程中，包含了潛伏和細胞溶解的過程。然而，從潛伏期過渡至細胞溶解期，病毒基因的調控表現並不清楚。第一部分的研究中，將使用TPA和HU兩種藥品加入EB病毒持續感染的B95-8細胞，用以分析EB病毒整體基因表現的情況。在加入TPA四十八小時後，EB病毒在細胞中的複製速率約增加二點五倍；而加入HU的情況，則降低EB病毒的複製速率達三分之二。大部分的EB病毒基因在細胞中的表現量均低於□型肌動蛋白。在加入TPA後，多數的病毒基因表現都會增加；相反的，當加入核甘酸合成抑制劑HU後，多數屬於細胞溶解期表現的基因會呈現明顯的抑制情況。在這部份的研究中，定量即時PCR技術的確是個可以用來偵測在藥物影響下EB病毒的基因表現。
在鼻咽癌切片檢查中，經常可以發現EB病毒第二A型潛伏膜蛋白(LMP2A)的存在。最近由鼻咽癌切片檢體所獲的cDNA微陣列研究中發現，第二A型潛伏膜蛋白與細胞中的尿嘧啶雙磷酸葡萄糖去氫酵素(UDP-glucose dehydrogease, UGDH)基因具有很高的相關性。尿嘧啶雙磷酸葡萄糖去氫酵素催化尿嘧啶雙磷酸葡萄糖(UDP-glucose)氧化反應變成尿嘧啶雙磷酸葡萄糖醛酸(UDP-glucuronic acid)，是構成細胞間質的前驅物。過量的細胞間質與眾多的細胞行為有關，其中包括了細胞的移行現象，因此尿嘧啶雙磷酸葡萄糖去氫酵素在研究癌細胞轉移上可能是個很好的研究對象。第二部份的研究，延續這個發現並證實：在EB病毒第二A型潛伏膜蛋白過量表現的HEK293細胞中，尿嘧啶雙磷酸葡萄糖去氫酵素基因其轉錄、轉譯功能及酵素活性都呈現增加情形，而細胞中的葡萄氨聚糖(glycoaminoglycans)含量也隨之增加。EB病毒第二A型潛伏膜蛋白所調控的尿嘧啶雙磷酸葡萄糖去氫酵素基因的啟動子區域，是位於其上游630 □ 486 bp，在這個區域的Sp1位點發生突變後，將減低EB病毒第二A型潛伏膜蛋白對其調控的影響。同時，加入Sp1的特定抑制劑與siRNA均可抑制由EB病毒第二A型潛伏膜蛋白所誘導的細胞移行現象。進一步尋找EB病毒第二A型潛伏膜蛋白調控尿嘧啶雙磷酸葡萄糖去氫酵素基因表現的訊息調控路徑，在加入特定抑制劑後，發現PI3K/Akt和ERK參與其中。這部份實驗提供了EB病毒第二A型潛伏膜蛋白調控尿嘧啶雙磷酸葡萄糖去氫酵素基因表現可能的分子調控機制。
在第三個研究部分，以siRNA降低人類腸癌細胞HCT-8中的尿嘧啶雙磷酸葡萄糖去氫酵素，發現在RNA和蛋白質表現上均有明顯的抑制。同時，在單層細胞培養中，可測量到葡萄氨聚糖的產量降低；在三維細胞培養中，延緩細胞球小體(spheroid)的培養時間，亦可破壞癌細胞的移行能力。因此，尿嘧啶雙磷酸葡萄糖去氫酵素或許亦可作為治療腸癌上的標的。

並列關鍵字

Epstein-Barr Virus ； UDP-glucose dehydrogenase ； latent membrane protein 2A ； gene expression ； cell migration

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