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系統識別號 U0026-1507201014545500
論文名稱(中文) 探討新穎癌症/睪丸抗原蛋白STK31之功能
論文名稱(英文) Characterization of a Novel Cancer/Testis Antigen, Serine/Threonine Kinase 31, and its role in Cancer Development
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 98
學期 2
出版年 99
研究生(中文) 謝昀容
研究生(英文) Christine Chin-jung Hsieh
學號 t3697404
學位類別 碩士
語文別 英文
論文頁數 76頁
口試委員 指導教授-郭保麟
指導教授-洪良宜
口試委員-黃溫雅
口試委員-唐堂
口試委員-洪義人
中文關鍵字 蛋白激酶  中心體  紡錘絲  癌症/睪丸抗原  男性不孕症  癌症治療 
英文關鍵字 protein kinases  centrosome  bipolar spindle  CT antigen  male infertility  cancer therapy 
學科別分類
中文摘要 許多的絲胺酸/蘇胺酸蛋白激酶已知與細胞週期檢查點功能的調控有關,在這些蛋白激酶中絲胺酸/蘇胺酸蛋白激酶31(serine/threonine kinase 31; STK31)為一功能未知的新穎基因。利用即時定量反轉錄聚合酶連鎖反應分析(real-time RT-PCR analysis),我們實驗室之前發現STK31只表現於睪丸組織,並且在造精功能缺陷病人的睪丸組織中,STK31 mRNA的表現量明顯地低於正常人。而最近的研究報告也指出STK31可被視為一癌症/睪丸抗原(CT antigen),並成為一個可能的癌症治療標的基因。實驗室進一步的研究則顯示STK31蛋白位在細胞中心體(centrosome)。基於這些發現,我們認為STK31可能參與調控細胞週期的進行。在我的論文研究中,我們發現在細胞週期進行過程中,STK31在所有時期,都會與中心粒周圍蛋白(pericentrin)共同表現並分布在中心體,除此之外在中期、後期及末期的階段STK31也會分布在染色體著絲點(centromere)、紡錘中央體(central spindle)及中體(midbody)上。同時我們也發現細胞內的STK31蛋白質的表現會隨著細胞週期的進行而有所變化。由於STK31蛋白位在細胞中心體(centrosome),細胞中心體也被稱為微管組織中心(microtubule organizing center; MTOC),因此我們也推測STK31可能參與了微管的聚合(microtubule assembly)。為了證實我們的推論,我們利用低溫的處理並觀察到抑制STK31的表現會導致微管的重組受到影響,並因而延遲了分裂期的進行並導致細胞分裂異常及細胞凋亡,而過量表現STK31同樣也會造成細胞週期異常及細胞的死亡。總結,我們對STK31這個新穎基因的研究結果發現其作用可能是參與細胞週期的調控,並與癌症的發生可能有關,因此未來也可能可以成為癌症治療的標的基因。
英文摘要 Many serine/threonine kinase proteins have been implicated in cell-cycle regulation. Among them, serine/threonine kinase 31 (STK31) is one of the novel kinase proteins of which biological functions remain largely unknown. Our preliminary results have previously shown that STK31 expression is limited to testis. Using immunofluroscence staining assay, we previously determined that STK31 localizes to the centrosome in GC-1 cells. A recent report has revealed that STK31 is overexpressed in cancer and may be considered as a novel cancer/testis antigen (CT antigen), which may serve as a cancer immunotherapy target. Based on these observations, we hypothesized that STK31 may involve in cell-cycle regulation in cancer development. In this study, we demonstrated that STK31 co-localizes with pericentrin in the centrosomal region throughout all phases of cell cycle. Interestingly, when cells undergo mitosis, STK31 also localizes to the centromeres during prophase and metaphase, and re-localizes to the central spindle during anaphase and concentrates at the midbody during telophase. Next, we found that the expression of STK31 varies throughout cell cycle and presumably is regulated through ubiquitin-proteasome degradation. Since STK31 was found to associate with the centrosome, we speculated that STK31 may take a part in microtubule assembly. Using cold treatment experiment, we verified that knockdown of STK31 would result in microtubule organizing defects. In addition, STK31 deficiency delayed the mitotic progression in GC-1 cells, and caused mitotic catastrophe and led to apoptosis in AZ521 cells. Overexpression of STK31 induced cell-cycle aberrations and eventually cell death. This study characterized a novel Ser/Thr kinase protein, which may participate in cell-cycle regulation during human cancer development. Therefore, it has a great potential for future therapeutic application in cancer therapy.
論文目次 中文摘要 i
ABSTRACT ii
ACKNOWLEDGEMENT iv
TABLE OF CONTENTS v
LIST OF FIGURES viii
LIST OF APPENDICES ix
ABBREVIATIONS x
1. INTRODUCTION 1
1.1 Cell cycle regulation 1
1.1.1 Cyclin-dependent kinases (CDKs): Key cell cycle regulators 1
1.1.2 Cell cycle progression 1
1.1.3 Cell-cycle checkpoints 2
1.2 Cell cycle kinases in cancer 3
1.2.1 Aurora kinase family 3
1.2.2 Polo-like kinase 1 4
1.2.3 Spindle assembly checkpoint proteins 5
1.2.4 Deregulation of cell cycle kinases in cancer 5
1.2.5 Targeting mitotic kinases in cancer therapy 6
1.3 The Centrosome 6
1.3.1 The function of the centrosome 6
1.3.2 The centrosomal proteins 7
1.3.3 Centrosome aberrations in human cancer 7
1.4 Serine/Threonine Kinase 31 (STK31) 8
1.4.1 Identification of STK31 in our lab 8
1.4.2 Current information of STK31 8
1.4.3 STK31 in human cancer 9
1.5 Cancer/testis antigen 10
2. OBJECTIVES OF THE PRESENT STUDY 11
3. MATERIALS AND METHODS 12
3.1 Cell culture 12
3.2 Western Blotting and antibodies 13
3.3 Total RNA extraction 15
3.4 Reverse transcription polymerase chain reaction 16
3.5 Real-Time RT-PCR 18
3.6 Immunofluorescence staining 20
3.7 Transient transfection of plasmids and shRNAs 21
3.8 Cold treatment 22
3.9 TUNEL assay 23
3.11 Time-lapse imaging 23
3.12 Lentivirus production 24
4. RESULTS 26
4.1 Differential expression of STK31 in various human cancer cell lines 26
4.2 Generation of STK31 monoclonal antibody 26
4.3 Subcellular localization of endogenous STK31 during mitosis 26
4.4 Centrosomal localization of exogenous STK31 throughout cell cycle 27
4.5 Co-localization of STK31 with mitotic kinases 28
4.6 Cell cycle expression of STK31 28
4.7 Knockdown efficiency of STK31 shRNAs 29
4.8 Lentiviral production of STK31 shRNAs and efficiency 30
4.9 Depletion of STK31 causes microtubule assembly defect during interphase 30
4.10 Depletion of STK31 resulted in mitotic progression delay in GC-1 cells 31
4.11 Depletion of STK31 causes mitotic catastrophe and leads to apoptosis in AZ521 cells 31
4.12 Exogenous overexpression of STK31 caused cell cycle aberrations 32
4.13 STK31 is overexpressed in clinical colorectal cancer specimens 33
5. DISCUSSION 34
5.1 The potential roles of STK31 in human cancer development 34
5.1.1 STK31 as a potential early diagnostic marker 34
5.1.2 STK31 may play a role in microtubule nucleation 34
5.1.3 STK31 may involve in SAC pathway 35
5.1.4 STK31 in tumorigenesis, cause or consequence? 36
5.2 Involvement of STK31 in spermatogenesis 37
5.2.1 STK31 may act as a cell cycle checkpoint in spermatogenesis 37
5.2.2 The potential roles of STK31 in maintenance of male germ-line stem cell population 38
5.3 Future perspectives on therapeutic values of STK31 39
REFERENCES 40
FIGURES 44
APPENDICES 65

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