2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/642616摘要：本計畫的目標是要了解類黏液蛋白 MUC15 如何調控腫瘤進程與發生。黏液蛋白在許多不同的癌細胞中扮演重要的角色，包括：細胞黏附(adhesion)、爬行(migration)、侵入(invasion)以及轉移等特性。細胞表面的黏液蛋白(membrane-bound mucins)可透過與其配體的結合或與其他受體的交互作用而進行訊息傳遞。最近我們發現MUC15 的mRNA 與蛋白過量表現於腫瘤組織中。在大腸癌細胞HCT116 暫時性的過量表現MUC15，增強了細胞的增生、黏附及侵入的特性，並且提升了ERK/MAPK 的磷酸化作用。因此，我們假設MUC15 能夠將訊息傳遞進入細胞，並因此調控癌細胞的行為。本計畫的特定目標為：1. 比較 MUC15 在正常組織與大腸直腸癌組織中的表現量，並分析得到的結果是否與大腸直腸癌的腫瘤進程與其他臨床特性有關。2. 分析 MUC15 在大腸癌細胞株中對細胞特性的各種影響。3. 在 SCID 小鼠動物模式中，分析MUC15 對腫瘤生長、轉移以及血管新生的影響。4. 分析 MUC15 影響細胞各種特性的分子機制。我們將生產高品質 MUC15 的單株及多株抗體。以real-time RT-PCR 和Western blot 分析MUC15 的mRNA 及protein 表現量，並以immunohistochemistry 呈現MUC15 在組織中表現的情形。藉由將大腸癌細胞株HCT116 和LoVo 的MUC15 基因knock-in 及knock-out 的技術，分析MUC15在細胞中的生物功能，此生物功能包括：凋亡、增生、群聚形成(colonyformation)、細胞週期、黏附、移行及侵入。另外，我們將以SCID 小鼠動物模式研究MUC15 對於腫瘤生長、轉移以及血管新生的影響。我們將藉由各種生化分析及微陣列(mircoarray)探討其分子機制。本計畫將使我們了解新型類黏液蛋白 MUC15 對於癌細胞行為的影響。此外，MUC15 影響癌細胞特性的分子機制也將被研究及揭曉。此研究的成果，將提供製藥工業發展新的診斷試劑及抗癌的藥物。<br> Abstract: The objective of this proposal is to understand how a novel surface mucin 15 (MUC15)regulates tumor progression and tumor development. Mucins have been suggested to play animportant role in cell proliferation, adhesion, migration, invasion, and metastasis in varioustypes of cancers. Transmembrane mucins also engage in signal transduction throughextracellular domain-mediated ligand binding or by interacting with surface receptors.Recently, we found that the expression of MUC15 mRNA and protein were elevated inprimary colorectal tissues compared with their normal counterparts by real-time RT-PCR.Transient over-expression of MUC15 in HCT116 colon cancer cells enhanced cellproliferation, adhesion, and invasion, as well as ERK/MAPK phosphorylation. Therefore, wehypothesize that MUC15 can transmit signals into cells and in turn regulate cancer cellbehaviors.Our specific aims are:1. to compare expression levels of MUC15 between normal and cancerous colorectaltissues and the obtained results will be correlated with tumor progression and clinicalcharacteristics of colorectal cancer patients.2. to investigate the effects of MUC15 on cellular properties of colon cancer cell lines.3. to analyze effects of MUC15 on tumor growth and metastasis as well as angiogenesis invivo by SCID mice animal models.4. to examine the molecular mechanisms by which MUC15 modulates cellular properties.We will generate high-quality polyclonal and monoclonal antibodies against MUC15.Expression levels of MUC15 mRNA and protein in paired primary colorectal tissues willbe analyzed by real-time RT-PCR and Western blotting. The tissue expression patterns ofMUC15 will be revealed by immunohistochemistry. The cellular functions of MUC15 invitro will be investigated in colon cancer cell lines HCT116 and LoVo by gene knock-inand knock-down techniques. To know the effects of MUC15 on colon cancer cells,cell-based assays, including cell proliferation, cell cycle, apoptosis, colony formation,migration, and invasion. Effects of MUC15 on tumor growth and metastasis as well asangiogenesis in vivo will be done in SCID mouse models. The underlying mechanisms bywhich MUC15 regulates cancer cell properties will be analyzed by general biochemistrytechniques and microarrays.The project will allow us to understand the effects of a novel mucin molecule on cancercell behaviors. In addition, the molecular mechanisms will be studied and unraveled. Theinformation gained by this study will provide pharmaceuticals to develop new diagnosticreagents and anti-cancer drugs in the future.