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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/68189


    Title: 4-aminobiphenyl誘導HepG2細胞中的microRNAs表現 並藉由microRNAs調控DNA修復機制;MicroRNA regulation of DNA repair gene expression in4-aminobiphenyl-treated HepG2 cells
    Authors: 邱炳豪;Chiou,Bin-Hao
    Contributors: 生命科學系
    Keywords: 4-胺基聯苯;氧化壓力;miR-513a-5p;XRCC2;DNA損傷;4-aminobiphenyl;oxidative stress;miR-513a-5p;XRCC2;DNA damage
    Date: 2015-07-29
    Issue Date: 2015-09-23 10:52:34 (UTC+8)
    Publisher: 國立中央大學
    Abstract: 流行病學研究指出,當生物體長期暴露在4-胺基聯苯 (4-aminobiphenyl,4-ABP) 的環境下,會造成肝臟細胞的DNA damage與DNA adducts,甚至會誘導膀胱癌的形成。4-ABP進入到人體後,會藉由肝臟細胞的phase I與phase II的氧化還原進行代謝,但過程中會產生自由基,進一步地導致DNA 雙股斷裂。本篇研究主要深入探討,4-ABP所造成的DNA損傷,其細胞內的自體修復機制為什麼不能立即做修補,是不是還有其他因素來調控修復機制? 首先利用comet assay來觀察DNA damage程度,處理4-ABP (0-300M)後,發現4-ABP確實會造成HepG2 cell的DNA損傷。PCR array與miRNAs microarray結果亦顯示,有16個關於DNA repair相關的基因表現下降,27個miRNAs 表現差異高達三倍以上,其中miR-630與miR-513a-5p為最顯著的表現。因此想深入探討,其DNA repair相關基因的失調,是否因為miRNAs所調控。研究結果顯示,miR-630會target在RAD18與EXO1的3’UTR上、miR-513a-5p則會target在XRCC2與FANCG之3’UTR。利用質體轉染技術,讓細胞內的miR-630和miR-513a-5p大量表現或抑制,皆會影響RAD18、XRCC2與FANCG的蛋白質表現。有文獻指出,4-ABP最主要會造成HepG2 cell的氧化性基因損傷並導致DNA double strands break,因此以miR-513a-5p與XRCC2為本研究主軸。
    4-ABP (75M) 與自由基抑制劑NAC結合處理,發現4-ABP誘導ROS的產生,會使miR-513a-5p大量表現,並進一步地導致XRCC2蛋白表現失調。將XRCC2 蛋白大量表現,其DNA damage的程度也有顯著性的被修復。本研究證明了4-ABP、oxidative stress、miR-513a-5p、XRCC2與DNA damage之間的互相調控機制,並釐清4-ABP所造成的DNA damage,其DNA repair system為什麼不能立即修復。最後,本研究提供了一個4-ABP所造成肝臟細胞的氧化基因損傷之預防檢測與治療的方向。
    ;The study goal was to evaluate the effects of 4-aminobiphenyl (4-ABP) on DNA damage based on the regulation of miRNAs to suppress some reducing DNA-repair proteins in human HepG2 cells. In this study, we used comet assay to determine that 4-ABP (0–300 M) induces DNA damage in HepG2 cells after 24 hours. DNA damage signaling pathway-based PCR arrays were used to investigate differential expressed genes response to 4-ABP treatment. In parallel, the miRNAs array was revealed that the expression of 27 miRNAs in 4-ABP-treated cells was at least 3-fold higher than that in the control group. Of these 27 miRNAs, the most significant expression of miRNA-513a-5p and miRNA-630 was further validated by qRT-PCR, which was predicted to target to RAD18、XRCC2 and FANCG genes, respectively, via bioinformatic analysis. In addition, overexpression and knockdown of miRNA-630 and miRNA-513a-5p inversely regulate the expression of RAD18、XRCC2 and FANCG proteins expression levels. 
    We found that ROS production is crucial for 4-ABP-induced miR-513a-5p expression and this phenomenon would be abolished by ROS inhibitor N-acetylcysteine. XRCC2 overexpression that the extent of DNA damage will be eminently repaired as well. Based on these, we indicated the mechanism of 4-ABP→ROS→miR-513a-5p --| XRCC2→DNA damage, and provide a potential application for prevention and therapy in future.
    Appears in Collections:[生命科學研究所 ] 博碩士論文

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