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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭永銘 | |
| dc.contributor.author | Mow-Jung Feng | en |
| dc.contributor.author | 酆茂蓉 | zh_TW |
| dc.date.accessioned | 2021-06-15T03:54:03Z | - |
| dc.date.available | 2013-09-09 | |
| dc.date.copyright | 2010-09-09 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-06-30 | |
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Cold Spring Harb Perspect Biol 1, a001883. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44747 | - |
| dc.description.abstract | 經由DNA微陣列的分析，我們發現KIAA0101 (p15PAF) 過度表現在肝細胞癌中。KIAA0101是一個與增殖細胞核抗原(PCNA)結合的蛋白質，但其功能目前尚未明瞭。在本篇論文裡，我們發現KIAA0101只表現在細胞週期中的S期。降低細胞中KIAA0101的表現會抑制細胞週期由G1期進入S期的進行，並且抑制BrdU的嵌入。降低KIAA0101的表現會誘導p21的過度表現。為了進一步的了解KIAA0101功能的分子機制，我們利用酵母菌雙雜交系統，尋找與之進行交互作用的蛋白質。我們發現Rad23A可與KIAA0101結合。經由免疫沈澱分析法我們證明EGFP-tagged KIAA0101確實能夠與Rad23A交互結合。Rad23A是一個在核苷酸切除修復擔任要角的蛋白質，也暗示KIAA0101可能參與DNA修復。結果如同預期，細胞群落分析的結果顯示，KIAA0101表現的降低會使細胞對紫外光敏感增高。在紫外光照射之下也觀察到KIAA0101表現降低的細胞其DNA合成會減少。這些結果顯示，KIAA0101是一個多功能的與增殖細胞核抗原結合的蛋白質，並參與調節DNA合成與修復。 | zh_TW |
| dc.description.abstract | By microarray analysis, we identified KIAA0101 (p15PAF) as a gene overexpressed in hepatocellular carcinoma. KIAA0101 is a proliferating cell nuclear antigen (PCNA)-binding protein of unknown function. In this study, we found that KIAA0101 was expressed only in S phase of the cell cycle. Knockdown of KIAA0101 inhibited cell cycle progression from G1 to S phase and suppressed BrdU incorporation. Knockdown of KIAA0101 induced p21 overexpression. To further delineate the molecular mechanism for the function of KIAA0101 protein, the yeast two hybrid assay was used to identify the interacting proteins. Rad23A was identified as a putative KIAA0101-binding protein. Using the immunoprecipitation assay, we demonstrated that EGFP-tagged KIAA0101 interacted with Rad23A, confirming the interaction of KIAA0101 and Rad23A in vivo. Rad23A is a major player in nucleotide excision repair. This result suggests that KIAA0101 play a role in DNA damage repair. Consistent with this suggestion, colony formation assay showed that KIAA0101 depletion sensitized cells to UV. Moreover, DNA synthesis was decreased in cells with KIAA0101 knockdown under UV irradiation. These results suggest that KIAA0101 is a multifunctional PCNA-interacting protein, and play important roles in regulating DNA synthesis and repair. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T03:54:03Z (GMT). No. of bitstreams: 1 ntu-99-R97444004-1.pdf: 3456267 bytes, checksum: 3a347c78f93b3e7a695d0cd0dbdba378 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 口試委員審定書 I
誌謝 II 中文摘要 III ABSTRACT IV CONTENTS V 1. INTRODUCTION 1 1.1 HEPATOCELLULAR CARCINOMA 2 1.2 MICROARRAY 3 1.3 EXPRESSION OF KIAA0101 IN HCC AND OTHER TISSUES 3 1.4 KIAA0101 IS A PCNA ASSOCIATED FACTOR 4 1.5 KIAA0101 REVIEWS 6 1.6 THE MECHANISMS OF DNA REPAIR 7 1.7 PURPOSES OF STUDY 8 2. MATERIALS AND METHODS 10 2.1 VECTORS CONSTRUCTION 11 2.2 CELL CULTURE 11 2.3 RNA INTERFERENCE 11 2.4 RNAI KNOCKDOWN OF KIAA0101 12 2.5 RNA ISOLATION AND RT-PCR 12 2.6 CELL PROLIFERATION ASSAY 13 2.7 CELL CYCLE SYNCHRONIZATION 13 2.8 FLOW CYTOMETRY 13 2.9 IMMUNOFLUORESCENT STAINING 14 2.10 WESTERN BLOT 14 2.11 YEAST TWO HYBRID SYSTEM 15 2.12 IMMUNOPRECIPITATION 15 2.13 UV IRRADIATION TREATMENT 16 2.14 COLONY FORMATION ASSAY 16 3. RESULTS 17 3.1 KIAA0101 IS EXPRESSED IN S PHASE 18 3.2 KNOCKDOWN OF KIAA0101 REDUCED CELL PROLIFERATION 18 3.3 KNOCKDOWN OF KIAA0101 INHIBITS CELL CYCLE PROGRESSION FROM G1 TO S PHASE 18 3.4 KIAA0101 IS REQUIRED FOR S PHASE PROGRESSION 19 3.5 KIAA0101 FOCI APPEARED BEFORE THE BRDU INCORPORATION 20 3.6 REDUCED DNA SYNTHESIS IN THE KIAA0101-KNOCKDOWNED CELLS 20 3.7 CDK INHIBITOR P21 IS ELEVATED IN KIAA0101-KNOCKDOWNED CELLS 21 3.8 RAD23A WAS IDENTIFIED AS A PUTATIVE KIAA0101-BINDING PROTEIN USING A YEAST TWO HYBRID ASSAY 21 3.9 INTERACTIONS BETWEEN KIAA0101 AND RAD23A IN VIVO 23 3.10 KNOCKDOWN OF KIAA0101 SENSITIZED CELLS TO UV 23 3.11 XPC PROTEIN EXPRESSION DID NOT CHANGE IN KIAA0101-KNOCKDOWNED HELA CELLS 23 3.12 GLOBAL GENOME DNA DAMAGE REPAIR UPON UV IRRADIATION REQUIRES KIAA0101 FOR DNA SYNTHESIS 24 4. DISCUSSION 25 4.1 KIAA0101 IS REQUIRED FOR S PHASE PROGRESSION 26 4.2 RAD23A WAS IDENTIFIED AS A PUTATIVE KIAA0101-BINDING PROTEIN 28 4.3 INTERACTION OF RAD23A AND KIAA0101 IN VIVO 29 4.4 XPC PROTEIN EXPRESSION DID NOT CHANGE IN KIAA0101-KNOCKDOWNED CELLS 30 4.5 THE POTENTIAL ROLE OF KIAA0101 IN DNA REPAIR 31 5. TABLES AND FIGURES 34 TABLE 1. RESULTS OF YEAST TWO HYBRID SYSTEM 35 FIGURE 1. KIAA0101 IS EXPRESSED IN S PHASE. 36 FIGURE 2. KNOCKDOWN OF KIAA0101 REDUCED CELL PROLIFERATION. 37 FIGURE 3. THE KIAA0101 IS REQUIRED FOR S PHASE PROGRESSION. 38 FIGURE 4. KIAA0101 EXPRESSION AFTER RELEASING FROM THE THYMIDINE- APHIDICOLIN BLOCK IN HELA CELLS. 39 FIGURE 5. BRDU-FITC STAINING AFTER RELEASED FROM THE THYMIDINE-APHIDICOLIN SYNCHRONIZATION. 40 FIGURE 6. KIAA0101 EXPRESSED BEFORE THE ONSET OF DNA SYNTHESIS. 41 FIGURE 7. BRDU INCORPORATION CYTOGRAM. 42 FIGURE 8. THE EXPRESSION LEVEL OF CDK INHIBITOR P21 AND P16 AFTER KNOCK DOWN OF KIAA0101. 43 FIGURE 9. THE INTERACTION OF SPECIFIC DOMAINS OF RAD23A WITH KIAA0101 TESTED IN THE YEAST TWO-HYBRID SYSTEM. 45 FIGURE 10. RAD23A INTERACTS WITH KIAA0101 IN VIVO. 46 FIGURE 11. KNOCKDOWN OF KIAA0101 SENSITIZED CELLS TO UV. 48 FIGURE 12. WESTERN BLOT ANALYSIS THE EXPRESSION LEVEL OF THE RAD23A-BINDING PARTNER XPC. 49 FIGURE 13. DNA SYNTHESIS DECREASE WAS OBSERVED IN KIAA0101-KNOCKDOWNED CELLS UNDER UV IRRADIATION. 50 6. REFERENCES 51 | |
| dc.language.iso | en | |
| dc.title | KIAA0101在細胞週期及DNA修復之角色 | zh_TW |
| dc.title | The role of KIAA0101 in cell cycle progression and DNA repair. | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 許輝吉,周涵怡,張?仁,葉秀慧 | |
| dc.subject.keyword | 增殖細胞核抗原,修復,細胞週期,複製,紫外光, | zh_TW |
| dc.subject.keyword | KIAA0101,PCNA,repair,cell cycle,Rad23A,UV irradiation, | en |
| dc.relation.page | 56 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-06-30 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 病理學研究所 | zh_TW |
| 顯示於系所單位： | 病理學科所 | |
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