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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/74075


    Title: 4-Aminobiphenyl 調控 miR-630 抑制 RAD18 表現誘導 Hep3B 細胞產生氧化性 DNA 損傷;The microRNA-630 is involved in oxidative DNA damage in 4-aminobiphenyl-treated Hep3B cells via downregulation RAD18
    Authors: 王方宗;Wang, Fang-Zong
    Contributors: 生命科學系
    Keywords: 對胺基聯苯;氧化壓力;miR-630;DNA 損傷;DNA 修復;同源重組;4-Aminobiphenyl;Oxidative stress;miR-630;DNA damage;DNA repair;Homologous Recombination
    Date: 2017-06-15
    Issue Date: 2017-10-27 13:07:32 (UTC+8)
    Publisher: 國立中央大學
    Abstract: 4-胺基聯苯(4-aminobiphenyl;4-ABP)為多環芳香族的聯苯胺衍生物,在燃燒的香菸與汽車排放的煙霧中發現大量4-ABP 化合物,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,解毒過程會產生自由基,進一步地導致DNA 損傷。4-ABP 15分鐘生成ROS,Comet assay 24小時後會導致Hep3B Cells 發生DNA 損傷,且 -H2AX (DNA double stand break sensor protein) 表現增加,RAD18 表現降低。再者,已知4-ABP 可誘導 HepG2 cells 產生多種 miRNA,尤其以 miR-513a-5p 和 miR-630 表現量最顯著。因此,本篇研究將探討4-ABP造成氧化型DNA損傷,是否透過miR-513a-5p 和 miR-630的調控。利用生物資訊軟體TargetScan 7.1預測此二miRNA 之標的基因,可能為RAD18、FANCG及XRCC2,由qRT-PCR 證實在HepG2與Hep3B細胞中只有miR-630 大量表現,且確認RAD18 轉譯會受到miR-630的直接調控。大量表現miR-630 導致RAD18 表現降低。4-ABP 與N-acetyl-L-cysteine (NAC)(一種自由基抑制劑) 合併處理會抑制ROS的產生,miR-630 表現也隨之降低,故得知miR-630 為ROS dependent miRNA。大量表現RAD18後,細胞核中 -H2AX foci聚集顯著降低,4-ABP造成homologous recombination 效率降低,此外,4-ABP抑制RAD18後也會使RAD51 (homologous recombination repair protein)表現降低。本研究發現了4-ABP造成基因毒性是透過ROS-dependent miR-630 活化,抑制RAD18,接著降低 Homologous Recombination活性,導致損傷之DNA 無法修復。
    本研究首次證實4-ABP可造成肝臟細胞的氧化基因損傷之其中一種 ROS-dependent miRNA 機制。
    ;4-aminobiphenyl (4-ABP) is a well-recognized human carcinogen, and has been found in textile dyes, cooking oil fumes, diesel-exhaust particles and cigarette smoke. 4-ABP induces oxidative stress and DNA damage that are associated with hepatic gene toxicity. My research foused on the role of miRNA-630 on RAD18, a DNA-repair protein. Firstly, 4-ABP induced ROS generation and DNA damage in Hep3B cells. The amount of 4-ABP-induced DNA double strand breaks (DSB) in a concentration- and time-dependent by determining the expression of -H2AX (the biomarker of DSB) with the methods of Western blot and Immunofluorescence staining assay. Luciferase reporter assay further confirmed miR-630 specifically targeted the 3’UTR of RAD18; thereby inhibiting RAD18 gene expression. Pretreatment of 4-ABP-treated cells with 5 mM N-acetyl-I-cysteine (NAC) attenuated the levels of miR-630 suggest that miR-630 was a ROS- dependent miRNA in 4-ABP-treated cells. 4-ABP treatment also inhibited the activity of DNA double stand break repair. This study showed that 4-ABP induces ROS generation in Hep3B cell line, and then increased the expression of the oxidative stress response-associated with the involvement of miR-630 in DNA damage. Elevated miR-630 expression inhibits RAD18 expression, with subsequent inhibition of homologous recombination.
    Appears in Collections:[生命科學研究所 ] 博碩士論文

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