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 (0-300M)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 (75M) 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.
