尿路上皮癌是泌尿道最常見的上皮腫瘤，近年來由於基因體分析技術的快速發展，發現基因的變異對於尿路上皮癌的致癌性與侵襲性愈來愈重要，探討這些基因的變異可以提供更好的診斷性、預後性及預測治療反應的相關資訊。為了找尋和尿路上皮癌侵襲度有關的基因，我們運用生物資訊探勘技術來分析膀胱尿路上皮癌基因表現資料庫，初步發現ZSCAN4和腫瘤侵犯組織的深度有顯著相關，比起表淺侵犯的腫瘤，侵犯到肌肉層以上的腫瘤其ZSCAN4的表現量是較低的。過去在幹細胞的研究發現，ZSCAN4有降低DNA損害反應、延長端粒(telomere)長度及穩定基因體的功能，然而，它在腫瘤細胞上的功能仍未明。首先，我們利用免疫組織化學染色的方式來探討ZSCAN4在340個上泌尿道尿路上皮癌及295個膀胱尿路上皮癌病人的預後意義以及它和各種臨床病理指標的相關性，初步發現在上泌尿道尿路上皮癌中，ZSCAN4低度表現的腫瘤，它的侵犯深度較深、較容易有淋巴結及血管的侵犯，在膀胱尿路上皮癌也是類似的情形，ZSCAN4低度表現的腫瘤其侵犯深度較深、細胞形態分級較高、較容易有淋巴結及血管的侵犯，在存活率分析方面，低度表現ZSCAN4的病人，他們的疾病相關存活率與無轉移存活率都是較差的。在生物體外的實驗顯示，當細胞高度表現ZSCAN4時，會抑制細胞增生、移行及侵襲的能力，也會抑制血管新生，這些發現都支持ZSCAN4在尿路上皮癌是一個抑制腫瘤生長的角色。此外，關於分子機制方面，我們發現ZSCAN4和pyrimidine ribonucleotides de novo biosynthesis pathway、cleavage and polyadenylation of pre-mRNA pathway及DNA double-strand break repair by non-homologous end joining pathway可能有潛在的相關性。總結來說，ZSCAN4在尿路上皮癌是扮演著抑制腫瘤生長的角色，低度表現ZSCAN4的腫瘤有較強的侵襲度，在臨床的預後方面也是較差的。

Urothelial carcinoma (UC) is the most common epithelial malignancy in the urinary system. With the advance in genome-wide analyses, genetic alternations play an increasingly important role in carcinogenesis and aggressiveness of UC. These molecular alternations may provide better diagnostic, prognostic and/or predictive value than conventional pathological classification. Through analysis of gene expression profiles of urinary bladder urothelial carcinoma (UBUC) from publicly available GEO dataset (GSE31684), Zinc finger and SCAN domain containing 4 (ZSCAN4) was identified as a significant downregulated gene in muscle-invasive UBUCs (T2-T4) when compared with non-muscle-invasive UBUCs (Ta-T1). In embryonic stem cells, ZSCAN4 was responsible for attenuating the DNA damage response, improving genomic stability and promoting telomere elongation during reprogramming. However, little is known about its functional role in cancer. Initially, we performed immunohistochemical study to investigate the prognostic significance of ZSCAN4 and its association with various clinicopathological parameters in 340 upper urinary tract urothelial carcinomas (UTUCs) and 295 UBUCs. In UTUC, low expression of ZSCAN4 was significantly associated with advanced primary pT stage (P=0.011), increased nodal metastasis (P=0.002) and increased vascular invasion (P=0.019). In UBUC, low expression of ZSCAN4 was significantly correlated with advanced primary pT stage (P<0.001), increased nodal metastasis (P=0.001), high histological grade (P=0.003) and increased vascular invasion (P=0.003). In survival analysis, low expression of ZSCAN4 acted as an independent negative prognostic factor for disease-specific survival and metastasis-free survival both in UTUC and UBUC. In vitro study revealed overexpression of ZSCAN4 suppressed cell proliferation, migration and invasion abilities, as well as angiogenesis. These findings supported the tumor suppressor role of ZSCAN4 in UC. In further investigation of underlying molecular mechanism, we found that ZSCAN4 has potential interactions with regulators involved in pyrimidine ribonucleotides de novo biosynthesis pathway, cleavage and polyadenylation of pre-mRNA pathway, and DNA double-strand break repair by non-homologous end joining pathway. In summary, ZSCAN4 played a key tumor suppressor role in UC. Low expression of ZSCAN4 was significantly associated with aggressive phenotypes and predicted worse outcome in UC patients.

論文審定書…………………………………………………………….………….. i
論文公開授權書……………………………………………………………………ii
中文摘要…………………………………………………………….…………….. iii
英文摘要………………………………………..………………………………..... iv
Directory of Figures…….…………………..……………………………..…....... viii
Directory of Tables………………………………………………………….…….. ix
List of abbreviations…………………………………………………..…….…….. x
1. Introduction………….……………………………………………...................... 1
2. Materials and methods………………………………………………………...... 5
2.1 Data mining of publicly available transcriptome…………………………... 5
2.2 Patients and tumor samples……………………………………………........ 6
2.3 Cell lines……………………………………………………………………. 7
2.4 Immunohistochemistry and scoring…………………………….………....... 8
2.5 Real time RT-PCR…………………………………………………………... 8
2.6 Expression plasmids and establishment of stable clones……......................... 9
2.7 RNA interference……………………………………………………............ 10
2.8 Flow cytometry analysis………………………………………………...….. 10
2.9 Transwell migration and invasion assays……………………..…………..… 11
2.10 BrdU assay……………………………………………………………..... 12
2.11 Tube formation assay………………………………………………...….. 12
2.12 Immunoprecipitation (IP)……………………………………..…….…… 13
2.13 Protein digestion…………………………………………………..….….. 13
2.14 Liquid chromatography tandem mass spectrometry analysis (LC-MS/MS)…..............................................................................................................… 14
2.15 Statistical analyses……………………………………………………….. 15
3. Results………………………………………………………………….….…….. 16
3.1 ZSCAN4 is identified as a significant downregulated gene in muscle-invasive UBUCs (T2-T4) when compared with non-muscle-invasive UBUCs (Ta-T1) .………………………………………………………..……………….….......... 16
3.2 Low mRNA transcript level of ZSCAN4 predicts worse outcome in the UBUC transcriptome (GSE31684) ..…………………………………..………..….….......... 16
3.3 Low protein expression of ZSCAN4 is associated with advanced disease status in UTUC and UBUC……………………………………………………..…….......... 16
3.4 Low protein expression of ZSCAN4 predicts worse outcome in UTUC and UBUC…………………………………………………………….……..………........ 17
3.5 Overexpression of ZSCAN4 inhibits cell proliferation, migration, and invasion abilities of UC cells……………………………………………………………...…….19
3.6 Overexpression of ZSCAN4 causes G0/G1 phase cell cycle arrest in J82 cells…………………………………………………………………………...…….. 20
3.7 Overexpression of ZSCAN4 suppresses angiogenesis in UC cell lines…………………………………………………………………………….…… 20
3.8 ZSCAN4 has potential interactions with pyrimidine ribonucleotides de novo biosynthesis pathway, cleavage and polyadenylation of pre-mRNA pathway, and DNA double-strand break repair by non-homologous end joining pathway……….……. 21
4. Discussion…………………………………………………………………..….... 22
5. Conclusion………………………………………………………………..….….. 26
6. Figures………………………………………………………………..…………. 27
7. Tables………………………………………………………………….…….…... 38
8. References……………………………………………………………….……..... 51
9. Publications…..………………………………………………………….………. 58

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