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.
