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研究生： 田崇辰
Tian, Chung-Chen
論文名稱： AATK基因低表現在胰腺癌中受到miR-338的調控並與低存活率相關
Downregulation of AATK by miR-338 correlates with poor prognosis in pancreatic ductal adenocarcinoma
指導教授： 黃柏憲
Huang, Po-Hsien
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： 39
中文關鍵詞： 胰腺癌 、AATK 、miR-338
外文關鍵詞： pancreatic ductal adenocarcinoma, AATK, miR-338
相關次數： 點閱：105  下載：0 
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    • 胰臟管腺癌是一種很惡性的疾病。在美國，它是所有癌症死亡人數中的第四名。到目前為止，手術切除仍然是胰臟癌唯一可以痊癒的治療方法。所以，找到胰臟癌早期的診斷方式、了解腫瘤的生理機制、以及找到更好的治療方法可以說是迫在眉睫的。我們利用全基因組的甲基化掃描，發現apoptosis-associated tyrosine kinase (AATK)這個基因，在腫瘤中(相較於正常胰臟組織)有著不同的甲基化程度。AATK位於染色體上17q25.3，是個在口腔癌、子宮頸癌和乳癌中常會缺失的位置。不過到目前為止，AATK在胰臟癌中的角色還未知。為了偵測AATK在胰臟癌中的表現情形，我們利用qPCR在細胞株中偵測AATK的messenger RNA表現；利用IHC染色在組織中偵測AATK蛋白表現。染色的結果顯示AATK在83%的胰臟癌中失去蛋白表現；這類失去AATK表現的病人伴隨著較差的預後。DNA的甲基化分析顯示有一群的胰臟癌病人AATK高程度的甲基化，但並不完全解釋了AATK蛋白的低表現。在本篇研究中，我們找到了一個編碼在AATK基因下游intron中的miRNA-MIR338，它會產生兩個成熟的miR-338-3p與miR-338-5p。更多的分析顯示miR-338-3p與miR-338-5p有潛力可以和AATK的mRNA 3’-UTR部分做結合並調控它的表現。總結來說，我們的結果支持了AATK可能在胰臟癌的發展及進程中扮演重要的角色，而miR-338可以調控其表現。

    Pancreatic ductal adenocarcinoma (PDA) is a lethal disease and the 4th leading cause of cancer death in USA in 2015. At present, radical operation is the only curative treatment for PDA. Therefore, the early-detection of cancer cells, understanding of the pathomechanistic biology, and effective therapeutic intervention are of exceptionally high priority to improve the disease outcome. Using genome-wide DNA methylation scan of PDA, we identified a differentially methylated region that located in the intron 1 of Apoptosis Associated Tyrosine Kinase (AATK) gene on chromosome 17q25.3, a region frequently lost in oral, cervical, and breast cancers. So far, the role of AATK as well as its function in PDA remains unclear. To determine the expression of AATK in PDA, we performed the qPCR for its mRNA expression in PDA cell lines and the immunohistochemistry (IHC) staining for its protein expression in a tissue microarray. IHC staining analysis indicated that AATK expression was downregulated in 83% of PDA patients, and low expression of AATK in tumor tissues was correlated with poor patient prognosis. DNA methylation analysis indicated that a subgroup of PDA patients were regulated by hypermethylation of AATK, but this only moderately accounted for the loss of AATK protein expression. In this study, we identified that MIR338 was a microRNA gene located within a downstream intron of AATK and encoded two mature miRNAs miR-338-3p and miR-338-5p. Further analysis of both mature miRNAs suggested that miR-338-3p and miR-338-5p have the potential to target the 3’UTR of its host gene mRNA and to regulate its expression. In conclusion, our results support the hypothesis that AATK may play an important role in the development and progression of PDA, and miR-338 plays a role in regulating the expression of AATK.

    INTRODUCTION 1 1. Pancreatic ductal carcinoma (PDA) 1 2. DNA methylation 2 3. Apoptosis-associated tyrosine kinase (AATK) 3 4. microRNAs and their roles in cancer 4 5. microRNA-338 4 RATIONALE AND HYPOTHESIS 6 SPECIFIC AIMS 7 METHODS AND MATERIALS 8 1. Clinical samples 8 2. Immunohistochemistry (IHC) staining and scoring 8 3. Cell culture 9 4. siAATK and miRNA-338 mimic transfection 10 5. Cell viability assay (MTS method) 11 6. Immunofluorescence staining 11 7. Quantitative reverse transcriptase-realtime PCR (qRT-PCR) 12 8. Luciferase reporter assay 14 9. In situ hybridization (ISH) 14 RESULTS AND DISCUSSION 16 Identification of different methylation level on AATK gene by genome-wide methylation scanning 16 AATK loss in PDA patients and associated with prognosis 16 AATK did not affect the cell viability and proliferation 17 miR-338 regulates the expression of AATK in parallel with first exon methylation 17 Discussion 18 CONCLUSION 22 REFERENCE 23 LIST OF FIGURES Figure 1. Hypermethylation of AATK in PDA tissues. 29 Figure 2. Expression of AATK is down-regulated in PDA. 31 Figure 3. Expression of AATK and miR-338 in pancreatic cancer cells and HPDE. 32 Figure 4. Cell viability was measured by MTS assay in si-AATK cells 33 Figure 5. Three perspectives of MiaPaCA-2 knockdown AATK, AATK (green), Ki67 (red) and DAPI (blue) 35 Figure 6. AATK expression was measured by qRT-PCR in miR-338-3p overexpress cells. 36 Figure 7. AATK expression was measured by qRT-PCR in miR-338-5p overexpress cells. 37 Figure 8. Luciferase reporter assay for miR-338-3p target AATK. 38 Figure 9. miR-338-3p expression was stained with ISH in PDA tissues. 39

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