膀胱尿路上皮癌(Urinary bladder urothelial carcinoma, UBUC)是一種常見的泌尿生殖惡性腫瘤，具有高死亡率和高治療成本。在我們過去的研究證明Epithelial membrane protein 2 (EMP2)在膀胱癌中扮演腫瘤抑制基因的角色，然而到目前為止EMP2在不同的癌症細胞中對細胞影響之確切的分子機制尚未釐清，因此在本研究中，我們將探討EMP2對於膀胱癌細胞株所誘發的細胞週期蛋白依賴激酶抑制劑(Cyclin-dependent kinase inhibitor, CKIs)的調控機制。為了詳細探討其分子調控機制，我們利用兩株膀胱癌細胞株分別過表現以及下調EMP2基因，觀察數個CKIs的表現。首先，在膀胱癌細胞中外源表現EMP2，會造成Transforming growth factor beta (TGFβ)以及數個CKIs的蛋白質和mRNA表現量上升，並且EMP2會增加SMAD2/3磷酸化進入細胞核進行轉錄調控。在細胞週期的部分，我們發現EMP2會誘導細胞週期停滯，在G2/M期增加及S期減少。另外，透過免疫共沉澱證明了EMP2會和purinergic receptor P2X 7 (P2RX7)交互作用。經流式細胞儀分析，EMP2和P2RX7會誘導細胞凋亡，並且當同時過量表現EMP2和P2RX7能夠發揮加乘效用。總結，根據本研究的結果，我們提出EMP2調控TGFβ增強從而導致細胞生長負向調控的詳細機制。證明EMP2透過TGFβR1，接著刺激磷酸化Smad2/3進入細胞核與Specificity protein 1 (SP1)共同賦予TGFβ誘導轉錄活性，上調CKIs使細胞週期阻滯，並且透過與P2RX7交互作用經由Caspase 8/9/3路徑誘導細胞凋亡。我們得出結論，EMP2參與細胞生長的調節，控制EMP2活性或表現是一個可行的潛在治療方式，作為新的腫瘤抑制基因。

Bladder urothelial carcinoma (Urinary bladder urothelial carcinoma, UBUC) is a common genitourinary cancer, with high mortality and high cost of treatment. Our previous studies have shown that Epithelial membrane protein 2 (EMP2) is a tumor suppressor gene in bladder cancer. However, in different types of cancer cells, the exact molecular mechanism of EMP2 induced cell cycle arrest and apoptosis was not clear. In this study, we will explore mechanisms of EMP2 induced Cyclin-dependent kinase inhibitor (CKIs) regulation in bladder cancer cell line. To investigate the molecular mechanisms in detail, we use two bladder cancer cell lines. Respectively, overexpression and knockdown EMP2 gene to observe the expression level of several CKIs. First, exogenous expression EMP2 gene in bladder cancer cells increased Transforming growth factor beta (TGFβ) and CKIs protein and mRNA levels, and EMP2 increased SMAD2/3 phosphorylation into nucleus leading to potentiating TGFβ-induced transcriptional. In the part of the cell cycle, we found EMP2 induces cell cycle arrest in G2/M phase and decreases in S phase. In addition, co-immunoprecipitation assay showed that EMP2 interaction with purinergic receptor P2X 7 (P2RX7). Flow cytometry analysis showed that EMP2 and P2RX7 induce apoptosis, and when co-transfected P2RX7 and EMP2, can play a synergistic utility of apoptosis. Summary, the results of this study, we propose the regulation of EMP2 enhanced TGFβ resulting in negative regulation of cell growth mechanisms in detail. Proof EMP2 mediated TGFβR1, then stimulate Smad2/3 phosphorylation into the nucleus and cooperate with Specificity protein 1 (SP1) confer TGFβ-induced transcriptional activity regulated CKIs cell cycle arrest and induced both extrinsic and intrinsic apoptosis through interaction with P2RX7. These results indicate that EMP2 acts as novel tumor suppressor regulating cell cycle and apoptosis. Therefore, controlling EMP2 activity or expression will be a viable potential tumor therapy.

論文審定書 i
致謝 ii
中文摘要 iii
英文摘要 iv
英文縮寫表 vi
圖次 vii
表次 viii
壹、緒論 (Introduction) 1
貳、材料與方法 (Materials and methods) 6
參、結果 (Results) 23
肆、討論 (Discussion) 44
伍、參考文獻 (References) 46

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