微西方墨點分析{Micro-Western Array (MWA)}是一種以抗體為基礎，且由GeSim Nanoplotter arrayer、 GE multiphor和 Licor Odyssey scanner組合而成的反向蛋白陣列分析儀器( reverse-phase protein array)。微西方墨點分析法(MWA)可同時檢測6~15個樣品在96~384個不同的抗體中，蛋白質或磷酸化的表現差異。在細胞系統的研究上，不論是蛋白質譜的表現特性或是訊號傳遞的網路，都已經被證實是可行的。我們使用微西方墨點分析法(MWA)去檢測708支不同的重要訊號蛋白，在正常攝護腺表皮細胞(RWPE-1, PZ-HPV-7)與攝護腺癌細胞(PC-3, DU-145, CA-HPV-10, LNCaP 104-S, LNCaP 104-R1)上的蛋白質表現差異。其中，我們發現一共有28支抗體在蛋白質的表現量上有著顯著的差異。在傳統西方墨點分析法驗證後，我們發現其中的四支抗體，分別為PAK2, PICK1, TEC 和 COX1，在正常攝護腺表皮細胞與攝護腺癌細胞上之間的蛋白質表現量，特別有顯著的差異。我們對於cyclooxygenase-1 (COX-1) 特別感興趣，原因在於COX -1在蛋白質的表現量上，正常的攝護腺表皮細胞都顯著的多於攝護腺癌細胞。然而cyclooxygenase (COX)是一種從花生四烯酸( arachidonic acid)到攝護腺素( prostaglandins)的生物途徑中，重要的中心酵素之一。我們的初步結果表示，COX-1的不正常表現影響了細胞增殖能力，以及攝護腺癌細胞的遷移和侵襲能力。我們懷疑COX-1可能藉由發炎途徑調控了攝護腺癌細胞的生長和轉移能力。除此之外，我們的蛋白質譜的數據將對未來的藥物發展和攝護腺癌細胞訊號網路的研究上相信會有很大的幫助。

Micro-Western Array (MWA) is an antibody-based modified reverse phase array composes of a GeSim Nanoplotter arrayer, a GE multiphor, and a Licor Odyssey scanner. MWA allows detecting protein expression level or phosphorylation status change of 96-384 different antibodies in 6-15 samples simultaneously. It has been shown to be effective in characterizing the protein profile or signaling network in cell systems. We used MWA to assay the protein expression profile in normal human like prostate epithelial cell lines (RWPE-1, PZ-HPV-7) vs. malignant human prostate cancer (PCa) cell lines (LNCaP 104-S, LNCaP 104-R1, PC-3, DU-145, CA-HPV-10) with 708 antibodies detecting important signaling proteins in cells. According to our study, we found 28 proteins differently expressed in cancerous PCa cells as compared to normal prostate cells. Four of the proteins, the PAK2, PICK1, TEC, and COX1 showed significantly different between normal and cancer PCa cells and were rarely studied previously. We focused on examining the role of cyclooxygenase-1 (COX-1) in PCa cells. Protein level of COX1 in normal cell line is much higher than in PCa cell line. Cyclooxygenase (COX) is the central enzyme in the biosynthetic pathway to generate prostaglandins from arachidonic acid. We discovered that overexpression of COX-1 suppressed the cell proliferation, migration, and invasion of PCa cells. These observations indicated the possibility that COX-1 may be involved in the regulation of tumor growth and metastasis of PCa, possibly via regulation of inflammation pathways. We believe that our protein profiling database will be useful for drug discovery and signaling network study in PCa research.

Abstract………………………………………………..Ⅰ
摘要………………………………………………….Ⅲ
誌謝…………………………………………………….Ⅴ
Catalog……………………………………………………..Ⅷ
Introduction…………………………………………..1
Prostate and Prostate Cancer……………………………..1
Normal and prostate cancer cell lines…………………….2
Micro-Western Array……………………………………..4
Materials and methods………………………………...8
Reagent……………………………………………..……8
Cell Culture…………………………………………..8
Cell Proliferation Assay……………………………..…8
Western Blotting Analysis……………………………….9
Micro-Western Arrays……………………......................10
Protein over-expression…………………………………13
siRNA knockdown of PAK2、PICK1 and TEC……………………………………………………...14
Wound Healing Assay……………………………….….15
Transwell Migration Assay…………………………..…15
Transwell Invasion Assay………………………….…16
Data Analysis…………………………………………17
Result and Discussion…………………………………20
Profiling the protein expression in human normal and cancerous prostate cell lines by Micro-Western Array (MWA)………….............................................................20
Differential protein expression between normal and cancer cell lines…………… ………………………………..…20
Traditional western blotting validated significant change protein in MWA………………………………………...21
Cell proliferation for knock down PAK2、PICK1 and TEC in LNCaP 104-R1……………………………………....23
PC3 over-express COX1 affected cell proliferation………………………………………….....26
Overexpression of COX1 in PC3 cells affected cell motility…………………………………………….……26
Overexpression of COX1 affected cell migration and invasion…………………………………………….…27
Over-expression level of COX1 protein affected some EMT markers……………………………………….…27
Over-expression level of COX1 protein affected some proliferation markers……………………………..…28
Figure………………………………………………………30
Figure 1.Micro-Western Array (MWA) is an antibody-based modified reverse phase array composes of a GeSim Nanoplotter arrayer, a GE multiphor, and a Licor Odyssey scanner………………………………30
Figure 2.Using high-throughput Micro-Western Array (MWA) to built up protein expression profile database……………………………………………….31
Figure 3.Raw data of RWPE1、PZ-HPV7 and CA-HPV10 by micro western blot…………………...32
Figure 4.Raw data of RWPE1、104S and 104R1 by micro western blot………………................................33
Figure 5.Raw data of RWPE1、Du145 and PC3 by micro western blot…………………………………………...34
Figure 6.Different protein expression level as assayed by MWA in the normal human prostate epithelial cell lines (RWPE-1, PZ-HPV-7) vs. malignant human prostate cancer cell lines (PC-3, DU-145, CA-HPV-10, LNCaP 104-S, LNCaP 104-R1) as shown by the heat map…………………… ……………………………35
Figure 7.Protein expression as determined by Micro Western Array (MWA) and conventional Western blot………………….. ………………………………36
Figure 8.Protein expression as determined by Micro Western Array (MWA) and conventional Western blot…………………...................................................37
Figure 9.Protein expression as determined by Micro Western Array (MWA) and conventional Western blot…………………. ………………………………38
Figure 10.The effect of over expression COX1 in PC3 on cellular proliferation……………………………..39
Figure 11.The effect of overexpression COX1 in PC3 on cell motility as determined by wound healing assay…………………….............................................40
Figure 12.Effects of over-expression of COX1 on migration and invas………………………………...…41
Figure 13.Overexpression of COX1 affected protein abundance of some EMT markers in PC-3 cells……………………………………………….42
Figure 14.Overexpression of COX1 affected protein abundance of some proliferation markers in PC-3 cells.……………………………………………..……43
Reference…………………………………………….……44
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