2012-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/646873摘要：大腸直腸癌近年來在全世界及臺灣的十大癌症死因中皆排名第三。雖然目前的研究逐漸了解多步驟的基因變化在大腸直腸致癌過程中所扮演的角色，但對此癌症的篩檢與預防，以及病人治療與預後的評估仍是一個重要的挑戰。在我們先前的研究中，利用分布在第四號染色體上的22 個微衛星標記，針對106 例大腸直腸腫瘤進行失異合性 (loss of heterozygosity, LOH) 分析，結果發現位於4q27 之D4S402 基因座的失異合性發生率最高，達32.9%。為了探究更精確的區域以尋找相關的抑癌基因，我們進一步以10 個微衛星標記建構4q25-4q28 區域(約12.9 Mb) 的高解析度刪除圖譜，定位出一個最小刪除區域位於4q28 (約3.51 Mb)。根據NCBI 資料庫顯示，此區域包含了9個假基因(pseudogenes)以及一個已知功能的基因，Protocadherin 10 (PCDH10)。最重要的是：PCDH10 基因發生基因刪除與大腸直腸癌的遠端轉移及病人總存活率有重要相關，因此我們假設PCDH10 基因即是存在於4q28 區域中與大腸直腸癌相關的抑癌基因。於是，我們先利用RT-PCR方法檢測12 株大腸直腸癌細胞株及10 對臨床腫瘤與其正常粘膜組織中PCDH10 基因的表現情形，結果顯示：在12 株大腸直腸癌細胞株中無法偵測到PCDH10 mRNA 的表現，8 例(80%)腫瘤則表現量明顯低於其正常粘膜組織。再利用real time RT-PCR 方法定量45 對臨床腫瘤與其正常粘膜組織中PCDH10 基因的表現，結果確定 37 例(82%)腫瘤之PCDH10 mRNA 表現量明顯低於其鄰近正常粘膜組織。此外，在抑癌功能研究方面，我們於大腸直腸癌細胞重新表現PCDH10 不僅可以降低細胞之增生、移行、侵犯和胞落形成能力，亦在體內小鼠模式中可以抑制腫瘤之生長。藉由以上的研究進展，本計畫中，我們將鑑定PCDH10 基因對於大腸直腸癌的抑癌功能，並探討其抑制腫瘤生成與肝臟轉移的分子機轉。本計畫研究目標如下：1. 藉由體外細胞模型及體內小鼠模型鑑定 PCDH10 的腫瘤抑制能力。2. 利用脾臟內注射模型探討 PCDH10 對於大腸直腸癌細胞之肝臟轉移的影響。3. 利用 cDNA microarray 分析探討PCDH10 抑制腫瘤生成及肝臟轉移的分子機轉。4. 藉由盲腸原位注射模型篩選具有高轉移能力的細胞株，以尋找與大腸直腸癌發生肝臟轉移的相關基因。5. 驗證 cDNA microarray 分析所得之候選基因，並據此建立細胞模型以探究PCDH10 抑癌相關的分子機制。<br> Abstract: Colorectal cancer (CRC) is the third leading cause of cancer-related mortality in the world and Taiwan. Colorectal carcinomas are characterized by the emergence of multiple chromosomal aberrations. By allelotyping with 22 polymorphic microsatellite markers spanning from chromosome 4pter to 4qter, we had defined the most frequent loss of heterozygosity locus, D4S402 (4q27), which occurred allelic imbalance in more than 32% of 106 colorectal tumors. Fine deletion mapping of 4q25-4q28 in thesecarcinomas further identified one common deletion region at 4q28. By searching with the NCBI database, there are 10 genes being posted at this region, including 9 pseudogenes and the only one gene, Protocadherin 10 (PCDH10), with known function. Importantly, allelic loss of PCDH10 gene was significantly associated with clinical outcome of CRC patients, including distant metastasis and poorer overall survival. Therefore, we have taken PCDH10 gene as the first priority to investigate its role of putative tumor suppressor gene (TSG) in CRC. We screened the mRNA expression of PCDH10 in 12 CRC cell lines and 10 pairs of tumor and normal mucosa tissues by semi-quantitative RT-PCR. The results showed PCDH10 mRNA was undetectable in all of 12 CRC cell lines, and was down-regulated in 8 of 10 (80%) tumor tissues compared with matched normal mucosa. To validate this finding, 45 pairs of CRC tissues were analyzed via real time RT-PCR, and PCDH10 expression was down-regulated in 37 (82%) colorectal carcinomas. Furthermore, for investigating tumor suppressor functions of PCDH10, the expression construct was established and then was transfected into CRC cells, HCT116. The preliminary results demonstrated that re-expression of PCDH10 in HCT116 cells not only inhibited cell proliferation, migration, invasion and anchorage-independent colony formation in vitro, but also suppressed tumor growth in vivo. With these progresses, in this project, we pursue to identify PCDH10 gene as a CRCassociated TSG and to explore the molecular mechanisms underlying PCDH10-mediated suppression of tumorigenesis and metastasis. Five specific aims are proposed as follows:Aim 1: To validate PCDH10-mediated tumor suppressor activities via in vitro cell models and in vivo xenograft tumor modelsAim 2: To investigate PCDH10-mediated suppression of liver metastasis via intrasplenic implantation tumor modelAim 3: To explore the molecular mechanisms underlying PCDH10-mediated tumor suppression via cDNA microarray analysisAim 4: To perform in vivo selection of highly metastatic cells via intracecal implantation tumor model to discover metastasis-associated genes in CRCAim 5: To validate the differential gene expression profiles and the molecular mechanisms of PCDH10-mediated tumor suppression大腸直腸癌腫瘤抑制基因分子機制Colorectal cancerTumor suppressor geneMolecular mechanism