異硫氰酸苄酯在膀胱癌中透過ERK/AP-1信息傳遞路徑增加抑癌基因miR-99a表現
陳宏恩1、陳栢均2、蔡德甫1、楊尚哲2、林宜佳1,4、仇光宇1、林致凡2、黃一勝1,2,3,4
1新光醫院 泌尿科、2新光醫院 中央研究室、3台北醫學大學 醫學院、4輔仁大學 醫學院
Benzyl isothiocyanate increases expression of miR-99a, a well-characterized tumor suppressor gene, in bladder cancer through ERK/AP-1 signal pathway
Hung-En Chen1#, Po-Chun Chen 2, Te-Fu Tsai 1, Shan-Che Yang2, Yi-Chia Lin1,4, Kuang-Yu Chou1, Ji-Fan Lin2, and Thomas I-Sheng Hwang1,2,3,4
Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital1, Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital2, Department of Urology, Taipei Medical University3, Division of Urology, School of Medicine, Fu-Jen Catholic University4, Taipei, Taiwan.
Purpose: miR-99a-5p, a well-established tumor suppressor, was down-regulated in cancerous bladder tissues. Benzyl isothiocyanate (BITC) is one of the isothiocyanate (ITCs) family, which exerts anti-cancer activity through apoptosis induction thus inhibiting malignant neoplasm. In this study, we investigated the expression pattern of miR-99a in BC cells treated with BITC, and defined the molecular mechanism by which regulated miR-99a expression.
Materials and Methods: The T24 and 5637 BC cells were treated with different concentractions of BITC for 24 h. The miR-99a expression level was confirmed by using miRNA RT-QPCR in 5637 and T24 cells. Meanwhile, the signal transduction which regulated miR-99a expression was further screened by using Western blot. Finally, the activation of AP-1 transcription factor was evaluated by using immunofluorescence staining and luciferase reporter assay.
Results: The BC cells treated with BITC dramatically increased miR-99a expression in a dose-dependent manner. The results also revealed that BITC activated ERK signal transduction, which was required for miR-99a expression in BC cells. We also found that AP-1 transcriptional activity was elevated by BITC treatment, and pretreatment with AP-1 inhibitor curcumin obviously reversed BITC-induced miR-99a expression in BC cells, suggesting that BITC promoted miR-99a expression through AP-1 activation in BC cells. Finally, pretreatment with ERK inhibitor U0126 abolished AP-1 activation, confirming that ERK/AP-1 signal cascade was necessary for miR-99a regulation in BC cells.
Conclusion: Treatment with BITC restored miR-99a expression, which was down-regulated while bladder cancer progression. Since miR-99a is a well-established tumor suppressor, BITC may serve as a novel therapeutic drug in bladder cancer treatment.
Key words: BITC, ERK, AP-1, miR-99a, bladder cancer