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研究生:林耕佑
研究生(外文):Ken-Yo Lin
論文名稱:甲基轉移酶基因FTSJ2在神經髓母細胞瘤與肝癌細胞株表現與功能之探討
論文名稱(外文):The study of methyltransferase FTSJ2 gene expression and function in Medulloblastoma and Hepatoma cell line
指導教授:陳全木陳全木引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:84
中文關鍵詞:FTSJ甲基轉移酶肝癌細胞神經髓母細胞瘤硫-腺苷酸甲硫胺酸甲硫胺酸代謝細胞生長
外文關鍵詞:FTSJmethyltransferaseSAMSAHHMethionine metabolismmigrationHepG2te671
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FTSJ2屬於大腸桿菌RNA核醣體次單元甲基轉移酶RrmJ直同源蛋白家族之一,其功能推測為將硫-腺苷酸甲硫胺酸(S-adenosylmethionine, SAM) 去甲基化轉化為硫-腺苷酸同胱胺酸(S-adenosylhomocysteine, SAH)並對RNA行甲基化修飾。生物體內SAM的來源則仰賴於甲硫胺酸代謝循環的供應,當甲硫胺酸代謝失調時,會造成細胞內SAM與SAH比率改變,進而調控相關基因表現並影響細胞的生長產生影響。本研究將豬FTSJ2基因轉殖於人類神經髓母細胞瘤細胞株te671(te671-FTSJ2)與肝癌細胞株HepG2(HepG2-FTSJ2),觀察FTSJ2大量表現對於甲硫胺酸代謝循環相關基因:甲硫胺酸腺苷酸轉移酶1A/2A(MAT1A/2A)、SAH水解酶(SAHH)、甲硫胺酸生成酶(MS)以及甜菜鹼-同胱胺酸甲基轉移酶(BHMT)的調控來評估對細胞內SAM /SAH比率是否具有影響以確認其甲基轉移酶的生物功能。結果發現HepG2轉殖FTSJ2提高了MAT1A的mRNA表現(p<0.05),並顯著降低了MAT2A的轉錄以及增加了SAHH的蛋白表現;te671-FTSJ2則呈現SAHH顯著的減少。SAHH的減少導致了te671-FTSJ2中F-actin表現受到抑制,造成細胞生長趨緩;免疫螢光染色的結果發現FTSJ2在粒線體中大量表現,說明了FTSJ2與其原核生物直同源蛋白祖先一般,會進入粒線體進行甲基化修飾,並調控胞內SAM/SAH比率進而影響粒線體轉錄作用;另外由te671與HepG2的比較可知,不同組織細胞間其甲硫胺酸代謝循環相關基因表現態樣與面對外來基因調控時反應並不是相同的。
FTSJ2 is a homologous protein of ribosomal RNA large subunit methyltransferase J (RrmJ) from bacteria. It may function as RrmJ that could catalyze S-adenosylhomocysteine (SAH) converted from demethylation of S-adenosylmethionine (SAM). Methionine adenosyltransferase (MAT) catalyzed the methionine and ATP to produce a principle biological methyl donor, SAM. There are two gene in MAT, MAT1A and MAT2A. It is known that the expression of MAT1A was level down in hepatocellular carcinoma (HCC), but MAT2A was increase. The MAT2A is also a negative control of intracellular SAM level. The functions of FTSJ2 are largely unknown, it is proposed that enhancement of FTSJ2 methyltransferase level may stimulate the MAT activity and SAH content in the cells. In this study, we overexpressed porcine FTSJ2 in HCC cell line HepG2 (G2-FTSJ2) and neuroblastoma cell line te671 (te671-FTSJ2) to prove the correlation between FTSJ2 and MAT. RT-PCR and Western blot were performed to analyze the expressions of MAT1a, MAT2a, SAH hydroase (SAHH), Methionine synthase (MS) and Batine-Homocysetine methyltransferase (BHMT) genes. Data showed that the expressions of MAT1A was 1.3 fold in G2-FTSJ2 than in HepG2 and Huh7. Contrary, MAT2A showed down regulation in G2-FTSJ2 than in HepG2 and Huh7 cell. Interestingly, overexpressed FTSJ2 in te671 cell decreased transcription of SAHH without affecting the RNA level of MAT1A and MAT2A. Our Results showed that FTSJ2 could participate in SAM metabolism cycle by affected the MAT and SAHH. In conclusion, the porcine FTSJ2 protein exhibits the potential function as methyltransferase in eukaryotic cells, especially responsible for abnormal methionine metabolism in HCC and neuroblastoma cell lines.
誌謝…………………………………………………………………………i
摘要…………………………………………………………………………ii
Abstract…………………………………………………………………….iii
表次……………………………………………………………………………vi
圖次……………………………………………………………………….......vii
壹、緒言………………………………………………………………….11
貳、文獻檢討………………………………………………………………12
一、 甲硫胺酸代謝循環與甲基化之關係…………………………………12
二、 已知甲硫胺酸代謝異常之相關疾病…………………………………18
三、 甲基轉化酶同源基因FTSJ2之介紹…………………………………24
四、 FTSJ2甲基轉化酶與甲硫胺酸代謝循環之探討.………………….32
五、FTSJ2基因於真核生物的功能性研究……………………………….35
叁、材料與方法…………………………………………………………………36
一、 組織之RNA萃取與cDNA之製備…………………………………36
二、 真核細胞豬隻FTSJ2表現質體pIRE-DsRED2-FTSJ2之建構……37
三、 肝癌細胞株HepG2與神經髓母細胞瘤細胞株te671來源與保存…44
四、 以細胞電穿孔法外源表現豬FTSJ2基因於細胞株HepG2及
te671…………………………………………………………………..45
五、外源FTSJ2表現偵測與甲硫胺酸代謝相關基因表現分析…………..46
六、細胞移動分析…………………………………………………………49
七、細胞侵襲(invasion)能力測試…………………………………………49
八、蛋白結構預測………………………………………………………50
九、北方墨點轉漬…………………………………………………………51
十、細胞免疫螢光染色……………………………………………………51
十一、統計分析……………………………………………………………52
肆、結果與討論……………………………………………………………53
一、FTSJ1與FTSJ2於人體組織表現之差異……………………………53
二、以RT-PCR分析FTSJ1與FTSJ2於肝癌細胞株之表現……………56
三、豬FTSJ2蛋白預測結構與人類內源FTSJ2蛋白結構比較…………56
四、轉染甲基轉移酶FTSJ2基因對細胞甲硫胺酸代謝調控的影響……58
(一) 反轉錄聚合反應分析FTSJ2轉殖對神經髓母細胞瘤
te671細胞株的影響……………………………….……59
(二) 反轉錄聚合鏈反應分析FTSJ2轉殖對HepG2肝癌細胞株的影響………………………………………………………………59
(三) FTSJ2轉殖對甲硫胺酸代謝循環相關蛋白之影響………….62
(四) 細胞免疫螢光染色分析FTSJ2之胞內分布………………….64
五、FTSJ2基因轉染對神經髓母細胞瘤te671細胞生長之影響………..67
六、FTSJ2調控SAHH表現量對F-actin之影響……………………………67
伍、結論……………………………………………………………………74
陸、參考文獻………………………………………………………………75
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