AITC 透過ROS 轉錄因子AP-1 調控mir-30a-5p 之表現
陳宏恩1、林致凡4、林宜佳1,3、蔡德甫1,3、仇光宇1,3、黃一勝1,2,3
1新光醫院 外科部 泌尿科; 2台北醫學大學醫學系 泌尿學科; 3輔仁大學醫學系 泌尿學科; 4新光醫院中央實驗室
Allyl isothiocyanate up-regulates miR-30a-5p expression through a ROS responsive transcription factor, activator protein 1 (AP-1)
Hung-En Chen1, Ji-Fan Lin4,Yi-Chia Lin1,3,Te-Fu Tsai1,3, and Kuang-Yu Chou1,3, Thomas I-Sheng Hwang1,2,3
1Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital, Department of Urology,
2Taipei Medical University, Division of Urology, 3School of Medicine, Fu-Jen Catholic University, 4Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
Background: Allyl isothiocyanate (AITC) is one of the most widely studied isothiocyanates that inhibits the survival of human prostate cancer (PCa) cells while not affecting normal prostate epithelial cells. microRNA 30a-5p (miR-30a-5p) was reported as a tumor suppressor and regulates epithelial to mesenchymal transition (EMT) in bone metastasis and castration-resistant PCa. In this study, we investigated whether AITC affects the expression of miR-30a-5p and the mechanism underlying this regulation.
Materials and Methods: Human PCa cell lines (Rv1 and PC3) were used in this study. The expression level of miR-30a-5p in AITC-treated cells was detected using stem-loop RT and QPCR. The expression of a validated miR-30a-5p target Ets Related Gene (ERG) by QPCR and Western blot in cells treated with AITC or transfected with miR-30a-5p. The expression level of phosphor-c-jun was detected by immunofluorescent and Westernblot. Reporter vector bearing antisense sequence of miR-30a-5p was served as inhibitor of AITC-induced miR-30a-5p.
Results: The expression of miR-30a-5p was increased in AITC-treated cells. The expression of ERG was down-regulated in AITC-treated or miR-30a-5p transfected cells. The expression of ERG was attenuated in AITC-treated cells transfected with miR-30a-5p antisense inhibitor, suggesting AITC regulates ERG expression through miR-30a-5p. Furthermore, we found the AP-1 is a potential regulator of miR-30a-5p expressionby analyzing 10 kb nucleotides sequence up-stream of miR-30a-5. Accumulation of c-Jun was detected in AITC-treated cells, and treatment of ROS inhibitor attenuated AITC-induced c-Jun activation, suggesting AITC activates c-Jun translocation was mediated by AITC-induced ROS. In addition, the up-regulated miR-30a-5p was also attenuated in AITC-treated cells pretreated with catalase further provide evidence that miR-30a-5p is an important effector of AITC.
Conclusions: Here, we show for the first time that AITC regulates the expression of miR-30a-5p through ROS generation and AP-1activation.These results could potentially contribute to a therapeutic application of AITC in prostate cancer patients.