ZC3H12D 是一種 CCCH 型鋅指蛋白質，屬於 MCPIP 蛋白質家族的一員。此家族蛋白質皆具有一個 CCCH zinc finger domain及 NYN domain，分別被認為具有 RNA 的結合及 RNase 功能相關。ZC3H12D 被報導為一新穎腫瘤抑制基因，其基因的突變在肺癌病人中具高發生率，且造成濾泡性淋巴瘤的惡化。近年發現 ZC3H12D 會抑制 JNK, ERK, NF-κB 訊息傳遞，且具有去泛素 (deubiquitination) 功能。本研究利用 Tet-on system 誘導 ZC3H12D 在 T-REx-293 細胞中的過度表現，結果發現 ZC3H12D 的過度表現會造成細胞存活率下降。由流式細胞儀分析發現 ZC3H12D 會造成細胞週期停滯於 G1 期及細胞凋亡。在分子機制方面，Akt 訊息傳遞路徑為調控細胞的增生、存活及細胞週期的進行最主要的路徑之一。ZC3H12D 的表現抑制 Akt 的 T308 與 S473磷酸化及其下游之訊息傳遞，如 FOXO1, Bad 及 GSK3β 的磷酸化，進而導致 Bcl-2/Bax 比例降低及 cyclin D 與 cyclin E 的降解，造成內因性細胞凋亡及細胞週期的停滯。近期研究發現，Akt 的活化需要經由Akt 泛素化 (ubiquitination)，使之移動至細胞膜進行磷酸化。而我們發現 ZC3H12D 會與 Akt 蛋白質互相結合，並且抑制 Akt 泛素化。本實驗結果顯示 ZC3H12D 可能藉由降低 Akt 泛素化而抑制其訊息傳遞，進而導致細胞週期停滯於 G1 期及細胞凋亡。

ZC3H12D is a CCCH-type zinc finger protein which belongs to MCPIP protein family. All MCPIP proteins contain a CCCH-type zinc finger domain and a NYN domain, which has been proposed with RNA-binding activity and RNase activity, respectively. ZC3H12D is reported as a novel tumor suppressor evidenced by high frequency mutation in lung cancer and transformed follicular lymphoma. Recently, ZC3H12D has been shown to inhibit toll-like receptor (TLR) 2 and TLR4-activated JNK, ERK and NF-κB signaling, and have deubiquitination activity. In this study, we found that overexpression of ZC3H12D by Tet-on system in T-REx-293 cells inhibited cell proliferation through inducing cell cycle arrest in G1 phase and apoptosis. Akt-mediated signal transduction is very important for cell proliferation, survival and tumorigenesis. Overexpression of ZC3H12D reduced the phosphorylation of Akt itself at T308 and S473 residues and its downstream signal molecules such as FOXO1, Bad, and GSK3β, and subsequently leaded to decrease of Bcl-2/Bax ratio, cyclin D and cyclin E expression. It was recently reported that ubiquitination of Akt is required for its membrane recruitment and activation. We found that ZC3H12D interacted with Akt and then deubiquitinated Akt. Taken together, these results suggest that ZC3H12D might induce G1 arrest and apoptosis through down-regulation of Akt ubiquitination.

致謝 I
中文摘要 II
Abstract III
縮寫表 (Abbreviations) 3
第一章 緒論 (Introduction) 5
壹、 ZC3H12D 簡介 5
貳、 ZC3H12D與腫瘤抑制之關係 7
參、 細胞週期 (Cell cycle) 9
肆、 細胞凋亡 (Apoptosis) 10
一、 內因性路徑 (Intrinsic Pathway) 11
二、 外因性路徑 (Extrinsic Pathway) 11
三、 執行路徑 (Execution Pathway) 12
伍、 Akt 訊息傳遞路徑 (Akt signaling pathway) 13
一、 Akt 訊息傳遞路徑調控細胞週期與細胞凋亡 14
二、 Akt 的泛素化調控 Akt 的活化 15
陸、 研究目的 (Study Aim) 16
第二章 材料與方法 (Materials and methods) 17
壹、 材料 17
一、 細胞株 18
二、 藥物及抗體 (Chemicals and Antibodies) 18
貳、 實驗方法 20
一、 細胞培養 (Cell Culture) 20
二、 MTT 呈色分析法 (MTT Assay) 21
三、 流式細胞分析儀測定 (Flow Cytometry Analysis) 22
四、 西方墨點法 (Western Blot) 23
五、 免疫沉澱法 (Immunoprecipitation) 25
六、 統計分析 (Statistical Analysis) 25
第三章 結果 (Results) 26
一、 過度表現 ZC3H12D 造成細胞存活率下降與細胞型態改變 26
二、 過度表現 ZC3H12D 導致細胞週期停滯於 G1 期及細胞凋亡 27
三、 過度表現 ZC3H12D抑制 Akt 的磷酸化及其下游之訊息傳遞 27
四、 過度表現 ZC3H12D 促進內因性細胞凋亡及 caspase-8 的表現 28
五、 過度表現 ZC3H12D 抑制 cyclin D 及 cyclin E 的表現 29
六、 過度表現 ZC3H12D 降低 Akt 的 ubiquitination 29
第四章 討論 (Discussion) 31
第五章 參考文獻 (References) 37
第六章 目次圖表 (Figures list) 41
Figure 1. Overexpression of ZC3H12D inhibited cell proliferation in T-REx-293 cells. 42
Figure 2. Overexpression of ZC3H12D induced G1-arrest and apoptosis in T-REx-293 cells. 45
Figure 3. Overexpression of ZC3H12D inhibited the activation of Akt and its downstream signal molecules. 46
Figure 4. Overexpression of ZC3H12D induced intrinsic apoptosis pathways. 48
Figure 5. Overexpression of ZC3H12D down-regulated the protein expression of G1 phase-related proteins. 49
Figure 6. Overexpression of ZC3H12D decreased Akt ubiquitination. 51
Figure 7. ZC3H12D might induce G1 arrest and apoptosis through the inhibition of Akt ubiquitination. 52

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