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研究生：	許瑋育 Hsu, Wei-Yu
論文名稱：	VAV1在啟動子與基因轉錄區之甲基化異常與胰臟癌轉移之關係 Aberrant Methylation of VAV1 Genebody and Promoter Associates with Pancreatic Cancer Metastasis
指導教授：	黃柏憲 Huang, Po-Hsien
共同指導教授:	陳昌熙 Chen, Chang-Shi
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	53
中文關鍵詞：	VAV1 、甲基化、免疫檢查基因
外文關鍵詞：	VAV1, methylation, immune checkpoint
相關次數：	點閱：81 下載：1
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胰臟癌在所有癌症當中為一個預後極差的癌症，五年均存活率約在百分之七以下。VAV1基因被證實和胰臟癌的低存活率有極大關連，但對於其異常表現及作用機制仍不明確。在此藉由一個高通量DNA甲基化定量系統，我們測得在胰臟癌病患於腫瘤檢體、胰液、癌症纖維細胞、周邊血單核球中，VAV1基因啟動子及基因轉錄區位置上的甲基化異常導致胰臟癌病患有較差的生存率。經由給予轉化生長因子β刺激後，於胰臟癌細胞株可發現有較高量VAV1基因表達，同時誘導產生片狀偽足。由於已知Kras基因和Smad4基因在調控胰臟癌進程扮演重要角色，因此當突變Kras基因或抑制SMAD4基因時，VAV1基因同時有過表達現象。此外VAV1基因的高度表達亦導致免疫檢查基因諸如：PD-L1,PD-L2,PD-1等過表達，進而使癌細胞逃避免疫監視，導致較惡化的病程。在本研究中我們發現，發現胰臟癌病患中VAV1基因和免疫檢查基因的甲基化表現量異常，同時突變的Kras基因使VAV1基因高度表達並活化免疫檢查基因PD-L1、PD-L2，使得癌細胞較為惡化。

VAV1 has been linked to poor survival of pancreatic cancer, but the regulatory mechanism leading to its ectopic expression in the clinical setting remains unclear. Using a high throughput quantitative DNA methylation assay, we identified that aberrant methylation patterns of VAV1 in the promoter and genebody correlated with poor prognosis of pancreatic ductal adenocarcinoma (PDAC) cases. By analyzing the cancer-associated fibroblasts (CAFs), pancreatic juice (PJ), and the peripheral blood mononuclear cells (PBMCs), aberrant VAV1 methylation identified a subset of pancreatic cancers. Transient transfection of VAV1 specific siRNA in PDAC cell lines PANC-04.03 revealed that the Transforming Growth Factor β (TGFβ) and the immune checkpoint pathway genes were differentially altered at the messenger RNA (mRNA) level. We firstly treated PDAC cell lines with TGFβ and identified that TGFβ induced the formation of lamellipodia structure in cells with high VAV1 expression. Furthermore, methylation analysis of candidate genes revealed that aberrant methylation of VAV1 and four immune checkpoint genes, including the programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) in the PBMC and the PDAC specimen. Our results showed that ectopic VAV1 expression associated with the immune check point genes in Kras mutant PDAC cell lines. In summary, our data indicate that TGFβ-VAV1 signaling plays an important role in promoting the progression, invasion, and evasion of immune surveillance in PDAC, which warrant further investigation of the use of TGFβ-targeted treatment in a subgroup of PDAC.

I. Introduction..................................................................................................1
1. Pancreatic cancer
2. DNA methylation
3. VAV1 gene
4. Transforming growth factor beta
5. Peripheral blood mononuclear cells 
6. Immune checkpoint gene
II. Rational and Hypothesis............................................................................4
III. Specific Aims.............................................................................................6
IV. Materials and Methods.............................................................................7
1. Cell line
2. EpiTyper Assay
  a. Bisulfite treatment
  b. Design primer
  c. Polymerase Chain Reaction (PCR)
  d. Shrimp Alkaline Phosphatase (SAP)
  e. In vitro transcription (IVT)
3. DNA/RNA/Protein purification
4. Reverse Transcription Polymerase Chain Reaction  (RT-PCR)
5. Real-time polymerase chain reaction (qPCR)
6. Plasmid purification
7. Transfection
8. Western-bloting
9. Immunofluorescence staining 
  a. Slide preparation
  b. Staining
  c. Image processing
V. Results........................................................................................................13
1. Aberrant methylation of VAV1 promoter and gene body regions is a tumor-specific 
  marker and is correlated with poor outcome.
2. Enhancer region plays an important role in VAV1 gene.
3. TGFβ treatment increase lamellipodia like phenotype in pancreatic cancer cell 
  lines.
4. VAV1 overexpress after transfection of Kras and knock down in SMAD4.
5. Methylation level of PBMC specimen in VAV1 gene in PBMC specimen was
  consistent confirm with that in the data with PDAC tissues.
6. Methylation level of immune checkpoint gene, CTLA-4, PD-L1, PD-L2 and PD-1
  have significance different between tumor and normal in PBMC specimens.
7. VAV1 mRNA level in PBMC specimen of pancreatic cancer patients separate in 
  two groups.
8. Kras mutation correlates with VAV1 overexpression and immune checkpoint gene 
  activation.

VI. Discussion.................................................................................................18
VII. Conclusion..............................................................................................20
VIII. Reference..............................................................................................21
VIIII. Figure and figure legend....................................................................24
Figure 1. VAV1 methylation level in promoter and gene-body.
Figure 2. Enhancer region methylation level in VAV1 gene.
Figure 3. TGF beta treatment in pancreatic cancer call lines.
Figure 4. Kras mutation and Smad4 loss of function associated with VAV1.
Figure 5. VAV1 methylation level in PBMCs specimen.
Figure 6. Methylation level of CTLA4 gene in PBMC specimen.
Figure 7. Methylation level of PDCD1 gene in PBMC specimen.
Figure 8. Methylation level of CD274 gene in PBMC specimen.
Figure 9. Methylation level of PDCD1LG2 gene in PBMC specimen.
Figure 10. VAV1 mRNA level of VAV1 in PBMCs specimen 
Figure 11. Overexpress and knock down VAV1 in pancreatic cancer cell line.
Figure 12. Correlation between VAV1, PD-L1 and PD-L2 genes.
Figure 13. VAV1coexpression with PD-L1 and PD-L2 genes.
Figure 14. mRNA level of VAV1, PD-L1 and PD-L2 genes.
Figure 15. Correlation between VAV1, Kras, PD-L1 and and PD-L2 genes.
X. Table...........................................................................................................50
XI. Appendix..................................................................................................51
A. PCR primer for EpiTYPER
B. PCR primer for q-RT-PCR
C. Antibody for Western-blot and immunofluorescence stain


                                    

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校內：2021-07-23公開
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