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研究生： 劉恬妤
Liu, Tien-Yu
論文名稱： 琥珀酸脫氫酶丙型過度表達在肺癌細胞中的影響
The effect of succinate dehydrogenase complex subunit C (SDHC) overexpression in lung cancer cells
指導教授： 張文粲
Chang, Wen-Tsan
學位類別： 碩士
Master
系所名稱： 醫學院 - 生物化學暨分子生物學研究所
Department of Biochemistry and Molecular Biology
論文出版年： 2020
畢業學年度： 108
語文別： 中文
論文頁數： 81
中文關鍵詞： 肺癌 、琥珀酸脫氫酶 、琥珀酸脫氫酶丙型 (SDHC) 、生物資訊資料庫 、SDHC過表達之肺癌細胞株
外文關鍵詞： lung cancer, succinate dehydrogenase, succinate dehydrogenase complex subunit C (SDHC), mega databases, SDHC overexpression
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    • 琥珀酸脫氫酶 (SDH) 在能量代謝中扮演重要角色，其同時參與檸檬酸循環 (TCA cycle) 以及電子傳遞鏈 (ETC)，其複合體為四個單元體SDHA、SDHB、SDHC以及SDHD並和四個輔因子所組成。先前本實驗室曾對肝癌的惡性程度與SDHB表達抑制之間的關係，然而發現同一SDH複合物的單元體SDHC，卻有不同的表達與作用，且對於多個大型數據庫進行分析，表明在肺癌之中當SDHC過表達，其病人預後較差。首先調查SDHC之研究背景，透過cBioPortal、Oncomine、Prognoscan以及COSMIC還有其他大型數據庫等的分析表明，其SDHC的高表達可增加癌細胞的生長，並且與SDH複合物的其他單元體結果不同，本實驗室已針對肺癌細胞株 (A549和H1299) 進行SDHC表達的抑制作用，發現此會導致癌細胞生長趨緩以及爬行能力的缺失。為了更進一步研究SDHC過表達在肺癌以及與肺癌惡性程度之間關係，利用已建構完成之pEGFP-N1-pSDHC及pSDHC，其過表達SDHC-GFP及SDHC之質體，進行轉染肺癌細胞 (A549和H1299)，建立穩定過表達SDHC肺癌細胞株。成功地在肺癌細胞中建立了SDHC過表達的穩定細胞株，並測定了對照組細胞(A549和H1299) 與SDHC過表達的肺癌細胞之間的差異，與對照組相比，其在正常情況下，利用MTT細胞生長測定、細胞遷移、傷口癒合細胞爬行測驗和產酸能力實驗，其結果沒有明顯的差異性。此外發現抑制SDHC表達時會誘導SDHA蛋白的上調，這與腫瘤細胞中SDHB抑制作用和SDHC過表達不同；發現SDHC過表達時，SDH複合物其他單元體SDHA及SDHB也會跟著上調，並推測其可能會發生互補情況，造成與對照組相比無明顯差異性。雖然SDHC過表達是否影響腫瘤的機制尚不清楚，但透過資料庫分析其SDHC的過度表達似乎是影響肺癌預後的重要因素。

    Succinate dehydrogenase (SDH) plays an important role in energy metabolism and participates in the tricarboxylic acid (TCA) cycle as well as the electron transport chain (ETC). It is a complex of four subunits, SDHA, SDHB, SDHC, SDHD, and four cofactors. Previously, our laboratory has established a relationship between tumor malignancy and the decrease of SDHB, a tumor suppressor, in liver cancer. Analysis of the multiple mega databases indicated that SDHC is overexpressed in lung cancer but has a poor prognosis. In addition, the SDHC and SDHB are subunits of the SDH complex but display different expression levels and effects. Analysis of the cBioPortal, Oncomine, Prognoscan, COSMIC, and other mega databases showed that high expression of SDHC increases cancer cell growth and that is different from other subunits of SDH complex. Additionally, knockdown of SDHC expression by shRNA molecules in the lung cancer cells H1299 and A549 resulted in the decrease of cell proliferation and crawling. To investigate further the effect of SDHC overexpression in lung cancer, the lung cancer cells were transfected with SDHC overexpression construct containing SDHC coding sequence fused with green fluorescent protein (GFP) gene, and only SDHC coding sequence. I established the stable cell line of the SDHC overexpression successfully in the lung cancer cells and assayed the difference between the control cancer cells (H1299 and A549) and the SDHC-overexpressed cancer cells. As compared with the control cancer cells, the SDHC-overexpressed cell lines exhibited that overexpression of the SDHC has no significant effects on MTT cell growth assay, cell migration, wound healing, and pH measurement under normal circumstances. In addition, knockdown of SDHC expression induced the up-regulation of SDHA proteins that were different from SDHB inhibition and SDHC overexpression in the tumor cells. We found that overexpression of the SDHC expression increased SDHA and SDHB expression because it is speculated that SDH subunits affect to be balanced off. Although the mechanism of SDHC overexpression affects the tumor is unknown. It seems SDHC overexpression is an important factor in lung cancer prognosis by analyzing the mega databases.

    中文摘要..................................................I 英文延伸摘要.............................................II 誌謝.....................................................V 目錄....................................................VI 圖表目錄 .............................................VIII 補充圖表目錄.............................................IX 第一章、緒論..............................................1 1-1 肺癌.............................................1 1-2 檸檬酸循環、電子傳遞鏈與細胞能量代謝................1 1-3 琥珀酸脫氫酶 (Succinate dehydrogenase，SDH).......3 1-4 琥珀酸脫氫酶 (SDH) 在癌症的研究....................4 1-5 生物資料庫的應用..................................5 1-6 研究目的..........................................6 第二章、實驗材料及方法.....................................7 2-1 實驗材料..........................................7 2-1-1 勝任細胞 (Competent cell).........................7 2-1-2 限制酶 (Restriction enzyme)......................7 2-1-3 細胞株...........................................7 2-1-4 化學藥品..........................................7 2-1-5 抗體............................................10 2-1-6 試劑............................................11 2-1-7 培養液..........................................11 2-1-8 緩衝液..........................................13 2-1-9 設備儀器.........................................16 2-1-10 實驗用的質體以及建構質體..........................17 2-2 實驗方法.........................................18 2-2-1 基本生物學技術...................................18 2-2-2 細胞培養程序.....................................22 2-2-3 細胞建立相關實驗.................................24 2-2-4 細胞分析相關實驗.................................25 2-2-5 統計分析.........................................27 第三章、實驗結果.........................................28 3-1 利用databases分析SDH各個subunits差異.............28 3-1-1 分析SDHA表現量在正常組織及腫瘤細胞之改變情形.......28 3-1-2 分析SDHB表現量在正常組織及腫瘤細胞之改變情形.......28 3-1-3 分析SDHC表現量在正常組織及腫瘤細胞之改變情形.......29 3-1-4 分析SDHD表現量在正常組織及腫瘤細胞之改變情形.......30 3-2 分析各種不同細胞株之SDHC的內生性表達以及挑選穩定過表達SDHC細胞株..............................................31 3-2-1 利用TMRM及Rodamine 123觀察SDHC-GFP蛋白位置.......32 3-2-2 過表達SDHC-GFP之H1299肺癌細胞株..................32 3-3 分析SDHC過表達對肺癌細胞株其SDH之其他單元體表達量..32 3-4 分析SDHC過表達對肺癌細胞生長與群落形成能力之影響...32 3-5 分析SDHC過表達對於肺癌細胞爬行能力之影響...........33 3-6 分析穩定過表達SDHC對於肺癌細胞糖解作用代謝之影響...34 第四章、討論.............................................35 4-1 挑選SDHC具有研究潛力目標.........................35 4-2 SDHC過表達與靜默株對肺癌細胞生長、群落形成能力及爬行能力之影響結果比較.........................................36 4-3 SDHC過表達與靜默株對於肺癌細胞糖解作用代謝之影響結果比較......................................................37 4-4 SDHC靜默株造成SDH其他單元體影響之探討.............37 4-5 SDHC過表達造成SDH其他單元體影響之探討.............37 第五章、結論.............................................40 第六章、參考文獻.........................................41 第七章、實驗結果圖表.....................................46 補充圖表.................................................76

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