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研究生：	黃詠翎 Huang, Young-Ling
論文名稱：	研究STK31在癌細胞中的功能及轉錄調控 Studying the functional role and transcriptional regulation of STK31 in cancer
指導教授：	郭保麟 Kuo, Pao-Link 洪良宜 Hung, Liang-Yi
學位類別：	碩士 Master
系所名稱：	醫學院 - 醫學檢驗生物技術學系 Department of Medical Laboratory Science and Biotechnology
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	52
中文關鍵詞：	絲胺酸/蘇胺酸蛋白激酶31 、癌症/睪丸抗原、細胞週期調控
外文關鍵詞：	serine/threonine kinase 31, cancer/testis antigen, cell-cycle regulation
相關次數：	點閱：121 下載：3
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絲胺酸/蘇胺酸蛋白激酶31(serine/threonine kinase 31；STK31)是一個新穎的癌症/睪丸抗原(CT antigen)，在正常組織中，除了睪丸之外，其他組織皆不會表現STK31。先前的報告指出，STK31在人類肝癌或是消化道相關的癌細胞中會表現。然而，對於STK31在癌細胞中所扮演的角色及其轉錄調控的機制至今仍未被釐清。為了進一步研究人類STK31的功能，我們製作可辨認人類STK31的多株抗體及構築會表現人類STK31的質體。利用免疫螢光技術，發現STK31位於細胞內的中心體及染色體上。西方墨點法及即時聚合酶連鎖反應，發現STK31蛋白質及mRNA的表現會隨著細胞週期的變化而改變。STK31在細胞分裂期表現量最低，並在細胞離開分裂期進入間期的G1 phase後，表現量又重新升高。這些結果暗示STK31蛋白可能參與調控細胞週期的進行有關。此外，在STK31的胺基酸序列上第644到652的位置，具有可能的destruction box （D-box）片段，此為泛素連接酶APC/C複合物辨認區塊（ubiquitin E3 ligase APC/C recognition motif），並且STK31的此區域在各物種間具高度保留性。將STK31蛋白在可能的的D-box區域突變，發現其在細胞週期的表現模式與野生型STK31不同。過度表現STK31並不影響細胞增生(proliferation)，但會促進細胞移行(migration)及侵襲(invasion)的能力。利用siRNA將降低癌細胞內STK31的表現，則會導致細胞凋亡。這些結果指出STK31對於癌細胞的生長與轉移，可能扮演相當重要的角色。另一方面，我們研究STK31的轉錄調控，發現相較於HeLa細胞，STK31 啟動子在AZ521細胞中具有較高的轉錄活性。STK31 啟動子的-326~-147位置為其主要的順式作用元件(cis-acting element)，此區間有多個具潛力的轉錄調控因子（trans-acting factors）結合位。實驗證實，ARNT及SP1為STK31重要的轉錄調控因子。

The serine/threonine kinase 31 (STK31) is a novel cancer/testis antigen (CT antigen) that restrictedly expresses in testis but not the normal tissues. The previous report indicated that human STK31 (hSTK31) is overexpressed in gastrointestinal and hepatic cancer. However, the physiological role and the transcriptional regulatory mechanism of hSTK31 in somatic cancer cells are still unclear. In order to investigate the role of hSTK31, the mammalian expression vectors of hSTK31 were constructed and the specific polyclonal antibodies against hSTK31 were generated. By immunoflourescence assay, hSTK31 located in the centrosomal and chromosomal regions. Western blot analysis and real-time-PCR indicated that the expression of hSTK31 was cell-cycle dependent. The lowest level of hSTK31 was at the onset of mitosis and elevated during mitosis exit and early G1 stage. These results implied the involvement of hSTK31 in cell cycle progression. In addition, a putative destruction box (D-box), the ubiquitin E3 ligase APC/C recognition motif, located between the amino acids 644 and 652 of hSTK31 and is evolutional conserved. D-box mutant of hSTK31 alters the cell cycle-dependent expression pattern. Furthermore, overexpression of hSTK31 enhances the cell migration and invasion abilities but did not affect the cell proliferation. hSTK31 knockdown by lantivirus-derived shRNA resulted in cell apoptosis. These results suggest that hSTK31 may play important roles in cancer cells. In addition, the promoter activity of hSTK31 in AZ521 cells is higher than Hela cell. A positive cis-acting element of hSTK31 promoter is located at the -326~-147, and both ARNT and SP1 are important transcriptional factors of hSTK31.

1.    Introduction....1
1.1   Cell cycle....1
1.1.1 Cell cycle stage....1
1.1.2 Cell cycle progression....1
1.2   Cell cycle kinase....2
1.2.1 Serine/threonine kinase....2
1.2.2 Polo-like kinase....3
1.2.3 Dysregulation of cell cycle kinase in cancer....4
1.3   Serine/threonine kinase 31 (STK31)....5
1.3.1 Identification of STK31....5
1.3.2 Previous information of STK31....5
1.3.3 STK31 in cell cycle regulation....6
1.4   Cancer/testis antigen....6
1.5   APC/C in cell cycle kinase degradation....7
2.    Materials and methods....9
3.    Result....17
3.1   Expression levels of hSTK31 are various in different 
         cancer cell lines....17
3.2   The expression of hSTK31 is cell cycle-dependent....17
3.3   The expression pattern of hSTK31 in cell cycle 
         progression may under the regulation of APC/C....19
3.4   Overexpression of hSTK31 not affect cell 
         proliferation ability and cell cycle distribution 
         but knockdown the expression of hSTK31 induces 
         cell apoptosis....20
3.5   Overexpression of hSTK31 promotes cell motility....21
3.6   Both ARNT and SP1 are the potential trans-acting 
         factors of hSTK31....22
4.    Discussion....24
4.1   The potential role of hSTK31 in cell cycle 
         progression....24
4.2   APC/C is the potential regulator for hSTK31....24
4.3   hSTK31 may involve in cancer malignancy....25
4.4   Expression of hSTK31 is regulated by ARNT and 
         SP1....26
5.    Reference....28
6.    Figures....31
      Figure 1. Expression level of endogenous hSTK31 mRNA 
         is different in various cancer cell lines....31
      Figure 2. Generation of the specific STK31 
         antibodies....32
      Figure 3. Subcellular localization of hSTK31....33
      Figure 4. hSTK31 is decrease at M phase and elevated 
         during mitotic exit....34
      Figure 5. Expression of endogenous hSTK31 protein is 
         cell cycle dependent....35
      Figure 6. hSTK31 protein expression is in a cell 
         cycle dependent manner....36
      Figure 7. The illustration indicates the expression 
         pattern of hSTK31....37
      Figure 8. Expression of hSTK31 is regulated by APC/C 
         ubiquitine machinery....38
      Figure 9. Overexpression of hSTK31 does not alter the 
         cell proliferation ability....39
      Figure 10. Overexpression of hSTK31 does not change 
         the cell cycle population....40
      Figure 11. Knockdown expression of hSTK31 results in 
         an obvious increase of  sub-G1 cell and decrease 
         G2/M cell population....41
      Figure 12. Knockdown hSTK31 not only leads to G1, S 
         and G2/M phase cell decreases but also obvious 
         increases sub-G1 cell....42
      Figure 13. Knockdown expression of hSTK31 increases 
         the expression of of active caspase 3....43
      Figure 14. Knockdown expression of hSTK31 results in 
         cell apoptosis....44
      Figure 15. Overexpression of hSTK31 increases cell 
         migration ability....45
      Figure 16. Overexpression of hSTK31 promotes cell 
         motility....46
      Figure 17. MMP2 mRNA expression level is influenced 
         by hSTK31....47
      Figure 18. Promoter activity of hSTK31 in AZ521 cells 
         is higher than HeLa cells....48
      Figure 19. The cis-acting element of hSTK31 promoter  
         is located at the -325~-146....49
      Figure 20. Both ARNT and SP1 are important 
         transcriptional factors for hSTK31....50
7.    Appendices....51
      Appendix 1. The cell cycle regulation....51
      Appendix 2. Schematic summary of spermatogenesis in 
         humans and cancer/testis (CT) antigen 
         expression....52

                                    

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校內：2015-02-07公開
校外：2015-02-07公開

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