利用蛋白質體學鑑定受 Valosin-containing protein 調控之宿主細胞因子

Abstract

摘要 胃癌是世界上最常見癌症中的第四名，也是所有癌症死亡率的第二名，全球每年至少有七十萬人死於此疾病。胃癌發生的原因很多，其中一個非常重要的因子乃幽門螺旋桿菌 (Helicobacter pylori, H. pylori)。近年來許多科學家致力於研究幽門螺旋桿菌導致胃癌的機制，使用的研究方法包括動物實驗、mRNA 微陣列技術 (mRNA microarray)、蛋白質體學技術 (proteomics approach) 等，希望能找出幽門螺旋桿菌感染引起的差異性表現基因或蛋白質，以進一步探討胃癌發生的分子機制。本實驗室利用蛋白質體學技術，比較感染幽門螺旋桿菌及未感染的 AGS 胃腺癌細胞，發現幽門螺旋桿菌感染後表現量會上升的蛋白質，有許多是可能導致癌症的因子，其中一個重要的蛋白質為 valosin-containing protein (VCP)。近年來發現 VCP 在許多不同的癌症都有大量表現的情形，且其在病人組織中的含量可作為癌症預後狀況的指標。然而 VCP 與癌症發展之間的關係目前尚未完全研究清楚。本論文的實驗目的是希望利用蛋白質體學技術，研究 AGS 胃腺癌細胞大量表現 VCP 後，會看到哪些差異性表現的蛋白質。 利用二維螢光差異凝膠電泳 (2-D DIGE) 分析，發現 VCP 大量表現後會增加表現的宿主蛋白質有 16 個，會減少表現的有 15 個，然後利用膠體內水解及串聯質譜儀鑑定這些蛋白質的身份；再配合本實驗室詹佳欣學姊利用 mRNA microarray 找到 VCP 大量表現造成的差異性表現基因；以及搜尋現有文獻；並根據西方墨點法，得到下列五點實驗結果：(1) 許多mRNA microarray 分析中表現量上升的基因為 NF-κB 的下游產物，且 p-IκBα 含量會下降 (西方墨點法)；(2) 利用西方墨點法發現抑制細胞週期的 p21、p27 及促進細胞凋亡的 p53 蛋白質量會下降，而促進細胞週期進行的 cyclin D1 會上升；(3) GADD34、GADD45基因表現量下降 (mRNA microarray)，以及 eIF4E (2-D DIGE)、c-Myc (西方墨點法) 蛋白質量上升；(4) keratin 19、cathepsin D (2-D DIGE)、Hif-1α (西方墨點法) 蛋白質含量上升；(5) 幽門螺旋桿菌促使 p-IκBα 含量下降以及 Hif-1α 含量上升的情形，在加入 VCP siRNA 後會消失。以上證據顯示 VCP 大量表現後，一方面促使細胞存活、生長與增殖，另一方面也會抑制細胞凋亡。由於文獻報導 VCP 對於 Akt 訊息傳遞是必要的，因此推測上述看到的現象乃因大量表現的 VCP 促進了 Akt 的下游訊息。 本篇論文結合 mRNA 微陣列及蛋白質體學技術，找到許多會受 VCP 調控之宿主細胞因子，並且由這些實驗結果，暗示了 Akt/VCP 的訊息傳導可能在幽門螺旋桿菌所誘導的胃癌機制中扮演一個重要的角色。未來經過更進一步實驗確認後，將可針對可能的 Akt 訊息傳導路徑來找尋抑制幽門螺旋桿菌導致之胃癌的方法。

Abstract Gastric cancer is the fourth most common cancer and the second most common cause of cancer-related death in the world — killing more than 700,000 people every year. There are many factors that lead to gastric cancer, one of which is infection by Helicobacter pylori (H .pylori). For the past few years, by utilizing proteomics approaches, mRNA microarray analyses, and animal model experiments, many scientists have devoted their efforts in studying the mechanisms of gastric cancer progression caused by H. pylori. By using proteomics approaches, our laboratory found that the protein expression level of many cancer-related factors were elevated in AGS cells after H. pylori infection, and one important factor was valosin-containing protein (VCP). It is found recently that VCP overexpresses in almost every kind of cancer, and the level of VCP in cancerous tissues is a prognosis marker for cancer diseases. The specific aim of this thesis is to investigate the differential-expressed proteins in AGS cells after overexpressing VCP by proteomics approaches. Sixteen proteins were up-regulated and fifteen proteins were down- regulated after VCP overexpression in 2-D DIGE analyses. According to LC-MS/MS identification, mRNA microarray analyses, literature search, and Western blot (WB) confirmation, we got the following five results: (1) many genes with increased expression level in mRNA microarray analyses were downstream products of the transcription factor NF-κB, and the protein level of p-IκBα was decreased (WB); (2) the protein level of cell cycle inhibitors, p21 and p27, and the proapoptotic factor p53 were decreased; on the other hand, the cell cycle promoting factor cyclin D1 was increased (WB); (3) the gene expression level of GADD34 and GADD45 (mRNA microarray) were decreased, and the protein level of eIF4E (2-D DIGE) and c-Myc (WB) were increased; (4) keratin 19, cathepsin D (2-D DIGE), and Hif-1α (WB) were up-regulated in protein level; (5) the phenomena of H. pylori-induced p-IκBα down-regulation and Hif-1α up-regulation were diminished after adding VCP siRNA. The aforesaid evidences indicated that VCP not only promoted AGS cell survival, growth and proliferation, but also inhibited apoptosis. VCP was documented to play an essential role in Akt downstream signaling, so we proposed the aforementioned findings were caused by the facilitation of Akt signaling in VCP-overexpressed cells. Combining mRNA microarray analyses and proteomics approaches, we have found many host factors regulated by VCP. According to the results, we proposed that Akt/VCP signaling might play an important role in H. pylori-caused gastric cancer.