降低KDM4C基因表現可抑制攝護腺癌細胞之生長、移動與侵襲

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研究生：	汪碧娟 Wang, Bi Juan
論文名稱：	降低KDM4C基因表現可抑制攝護腺癌細胞之生長、移動與侵襲 Knockdown of KDM4C suppresses the proliferation, migration, and invation of prostate cancer cells
指導教授：	楊孝德 Yang, Shiaw Der 褚志斌 Chuu, Chih Pin
口試委員:	汪宏達張中和
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	36
中文關鍵詞：	攝護腺癌、KDM4C
相關次數：	點閱：4 下載：0
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前列腺癌（Prostate cancer）在世界上最常見的癌症中排名第五。在台灣則是第六常見的癌症而且其發病率逐年增加。超過80%的前列腺癌患者死於骨轉移。賀爾蒙療法(Androgen ablation therapy)是治療轉移性前列腺癌的主要方法。然而，病患在接受賀爾蒙療法後1-3年會進展成去勢療法無效前列腺癌(castration - resistant prostate cancer，CRPC)，復發後病患的中位存活期為1-2年且目前並無有效的治療方法。KDM4組蛋白去甲基酵素是調控外基因(epigenetics)的重要酵素。其中，KDM4C被發現是雄激素受體AR (androgen receptor)的共同調節受體(co-regulator)，在CRPC中也發現KDM4C的表現量較高。雖然KDM4C在前列腺癌中所扮演的角色尚不明確，但在乳癌的研究中，KDM4C被認為是一個致癌基因(oncogene)。因此，本實驗利用siRNA抑制前列腺癌細胞LNCaP和C4-2B的KDM4C，結果發現前列腺癌細胞的增生、移動和侵襲能力明顯下降。進一步透過西方墨點法發現，當KDM4C表現量下降時，與EMT (epithelial-to-mesenchymal transition) 和Wnt signaling pathway相關的蛋白質表現量亦下降，像是Slug, Snail, vimentin and β-catenin。在異種移殖(Xenograft)原位模式(Orthotopic model)的動物實驗中也發現小鼠體內人類前列腺腫瘤的轉移能力受到抑制。我們的研究結果發現KDM4C能夠控制前列腺癌的發展與轉移，為KDM4C作為晚期前列腺癌治療標的提供了理論基礎。

Contents
Abstract ………………………………………………………………………………I
中文摘要…………………………………………………………………………….III
誌謝…. ………………………………………………………………………………IV
Contents…………………………………………………………………………... ..V
Figure contents…………………………………………………………………...… VII
1. Introduction …………………………………………………………….…………..1
2. Experimental aim and procedure………………………………………………… ...4
3. Materials and methods ……………………………………………………...……...5
3-1 Antibodies…………………………………………………………….……… .5
  3-2 Cell culture ………………………………………………………………...… ..5
3-2-1 Cell lines……………………………………………………………….. .5
3-2-2 Cell subculture……………………………………………………………..6
3-2-3 Cell thawing ………………………………………………………...…… .6
    3-2-4 Cell frozen ……………………………………………………….……… .7
3-3 siRNA transfection …………………………………………………..……… ...7
  3-4 Proliferation assay………………….………………………………..……….. ..8
  3-5 Transwell assay …………………………………………………………….…..9
  3-6 Wound healing assay………………………………………………………….10
3-7 Western blot analysis …………………………………………………….… ..10
    3-7-1 Protein extraction ……………………………………………...……… ...10
    3-7-2 Protein concentration measurement……………………………..…… ...11
3-7-3 SDS- page electrophoresis ………………………………… ………… ...11
3-7-4 Western blotting ………………………………………………………...12
  3-8 Orthotopic prostate cancer Tumorigenesis Model…………………………….13
3-9 Immunohistochemistry ……………………………………………………….14
3-10 Statistical Analysis…………………………………………………………...15
4. Results …………………………………………………………………………….16
4-1 Knockdown of KDM4C reduces prostate cancer cell proliferation…………...16
4-2 Knockdown of KDM4C suppresses the migration, invasion, and motility of prostate cancer cells……………………………………………………………17
4-3 Knockdown of KDM4C suppresses prostate tumor metastasis in nude mice……………………………………………………………………………18
4-4 Knockdown of KDM4C affected proteins involved in regulation of cancer cell metastasis……………………………………………………………………....19
5. Discussion ……………………………………………………………………… ..20
6. Figures……………………………………………… …………………………….23
7. References……………………………………………………………………….33

Figure Contents
Figure 1. KDM4C mRNA and protein expression levels in different prostate cells…23
Figure 2. KDM4C gene expression levels in prostate cancer patient samples………24
Figure 3. Knockdown of KDM4C reduced the proliferation of LNCaP and C4-2B 
cells………………………………………………………………………...25
Figure 4. Knockdown of KDM4C inhibited the migration and invasion of LNCaP and C4-2B cells as measured by transwell assay……………………………...26
Figure 5.Knockdown of KDM4C inhibited the migration of C4-2B cells as determined by wound healing assay………………………………………27
Figure 6. The efficiency of siRNA knockdown of KDM4C in C4-2B cells…………28
Figure 7. Hematoxylin and Eosin (H&E) stain of orthotopic model mice tissue……29
Figure 8. Immunohistochemistry (IHC) staining of nude mice prostate……………..30
Figure 9. Immunohistochemistry (IHC) staining of nude mice intestine…………….31
Figure 10. Knockdown of KDM4C in C4-2B cells affected expression levels of EMT marker and β-catenin proteins………………………………………………………..32
 

                                

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