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

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研究生：	潘韻如 Yun-Ru Pan
論文名稱：	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
指導教授：	張晃猷 Hwan-You Chang
口試委員:
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生命科學系 Department of Life Sciences
論文出版年：	2008
畢業學年度：	96
語文別：	英文
論文頁數：	98
中文關鍵詞：	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
外文關鍵詞：	Epstein-Barr Virus, UDP-glucose dehydrogenase, latent membrane protein 2A, gene expression, cell migration
相關次數：	點閱：2 下載：0
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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.

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。

Table of Contents

Table of Contents    II
Abstract (English)    V
Abstract (Chinese)    VII
Acknowledgement    IX
Abbreviations    X

Chapter 1  General Introduction    1

Chapter 2 Analysis of Epstein-Barr Virus Gene Expression upon Phorbol Ester and Hydroxyurea Treatment by Quantitative Real-Time PCR    7
2.1. Abstract    8
2.2. Introduction    9
2.3. Materials and Methods    11
2.3.1. Cell lines and growth conditions    11
2.3.2. Nucleic acid isolation and reverse transcription (RT) reaction    11
2.3.3. Conventional PCR Amplification of cDNA    12
2.3.4. Primer design    12
2.3.5. Conditions of qPCR and calculation of CT, dCT, ddCT, and dddCT values    12
2.4. Results     14
2.4.1. Effects of TPA and HU treatment on EBV genome copy number    14
2.4.2. Determination of primer specificity for qPCR analysis of EBV gene expression    14
2.4.3. Quality control of the template for the qPCR    15
2.4.4. Analysis of the relative abundance of EBV mRNA in B95-8 cells    16
2.4.5. Effects of TPA on EBV gene expression    17
2.4.6. Effects of HU treatment on EBV gene expression    18
2.4.7. Analysis of TPA treatment time and EBV gene expression    18
2.4.8. Effects of HU treatment time on EBV gene expression    19
2.5. Discussion    20
2.6. Tables    23
Table 2-1 dCT, ddCT and dddCT values of TPA treatment of EBV genes    23
Table 2-2 dCT, ddCT and dddCT values of HU treatment of EBV genes    25
2.7. Figures    27
Fig. 2-1. EBV genome copy numbers in a single B95-8 cell after TPA or HU treatment.    27
Fig. 2-2. Qualitative analysis of EBV mRNA between RT-negative and RT-positive pools.    28
Fig. 2-3. Relative mRNA abundance of EBV genes.     29
Fig. 2-4. Effects of TPA and HU on EBV gene expression.     30
Fig. 2-5. Effects of TPA- and HU-treatment for different time periods on the expression of EBV genes.     31

Chapter 3 Epstein-Barr virus latent membrane protein 2A upregulates UDP-glucose dehydrogenase gene expression via ERK and PI3K/Akt pathway    32
3.1. Abstract    33
3.2. Introduction    34
3.3. Materials and Methods    36
3.3.1. Cell lines and culture conditions    36
3.3.2. RNA extraction, reverse transcription (RT) reaction and quantitative real-time PCR (qPCR)    36
3.3.3. Measurement of UGDH activity    37
3.3.4. Quantitative measurement of glycosaminoglycans    37
3.3.5. Construction of promoter–reporter plasmids and site-directed mutagenesis    38
3.3.6. Transient transfection and luciferase assay    38
3.3.7. Western blotting analysis    39
3.3.8. Extraction of nuclear proteins and electrophoresis mobility shift assay (EMSA)    40
3.3.9. Three-dimensional collagen gel cell migration assay    41
3.4. Results    42
3.4.1. LMP2A upregulates UGDH gene expression    42
3.4.2. LMP2A increases GAGs production    42
3.4.3. The □632 to □486 region of UGDH promoter contains LMP2A responsive sequence    43
3.4.4. Identification of Sp1 binding motifs contributing to LMP2A-mediated UGDH promoter    44
3.4.5. Sp1 binds to the □563 to □555 region of UGDH promoter    45
3.4.6. Overexpression of Sp1 enhances UGDH promoter activation by LMP2A    45
3.4.7. Effects of Sp1 inhibitor and Sp1 siRNA on UGDH promoter activity    46
3.4.8. Involvement of Sp1 in LMP2A-mediated cell motility    47
3.4.9. LMP2A-mediated UGDH promoter regulation involves PI3K/Akt and ERK pathways    47
3.4.10. Identification of tyrosine residues in LMP2A essential for UGDH promoter activation    48
3.5. Discussion    50
3.6. Figures    54
Fig. 3-1. Effects of LMP2A on UGDH expression.    54
Fig. 3-2. Quantification of GAG in cells with or without LMP2A expression.    56
Fig. 3-3. Nucleotide sequence of the 5’-flanking region of the human UGDH gene.    57
Fig. 3-4. Deletion and site-specific mutation of UGDH promoter.    58
Fig. 3-5. Eletrophoretic mobility shift analysis (EMSA) of UGDH promoter.    59
Fig. 3-6. Determination of Sp1 effect on LMP2A-induced UGDH gene expression.    61
Fig. 3-7. Roles of Sp1 in LMP2A-induced cell motility in collagen gels.    63
Fig. 3-8. Identification of the signaling pathway responsible for LMP2A-mediated UGDH gene activation.     64
Fig. 3-9. Effects of LMP2A tyrosine mutations on human UGDH gene promoter activation.    66
Fig. 3-10. A model of UGDH gene activation by LMP2A.     67

Chapter 4 Migration of HCT-8 Colorectal Carcinoma Cells Is Impeded by Downregulating UDP-Glucose Dehydrogenase    68
4.1. Abstract    69
4.2. Introduction    70
4.3. Material and Methods    72
4.3.1. Cell lines, culture conditions, and transfection    72
4.3.2. RNA extraction, reverse transcription (RT) reaction and quantitative real-time PCR (qPCR)    72
4.3.3. Western bloting analysis    73
4.3.4. Quantitative measurement of glycosaminoglycans    73
4.3.5. Multicellular spheroid culture    74
4.3.6. Three-dimensional collagen gel cell migration assay    75
4.3.7. Transwell assay    75
4.4. Results    76
4.4.1. Suppression of human UGDH expression by siRNA    76
4.4.2. Decrease of GAG production by UGDH siRNA    76
4.4.3. UGDH down regulation resulted in delayed spheroid formation    77
4.4.4. Disruption of cell motilities of HCT-8 cells with UGDH siRNA effects    77
4.5. Discussion    79
4.6. Figures    81
Fig. 4-1. Effects of siRNA on UGDH expression.    81
Fig. 4-2. Effects of UGDH down regulation on glycoconjugate production in HCT-8 cell.    82
Fig. 4-3. Effects of UGDH down regulation on multicellular spheroid formation.    83
Fig. 4-4. Modulation of HCT-8 cell motility by UGDH siRNA.    84
Fig. 4-5. Reduction of cell motility by UGDH siRNA in the transwell assay.    85

References    86

Appendix    94

                                

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