Benzyl isothiocyanate promotes miR-99a expression through ERK/AP-1-dependent pathway in bladder cancer cells

Benzyl isothiocyanate (BITC), a bioactive natural product present in cruciferous vegetables, has been proved to prevent cancer progression through various mechanisms. In our previous report, we proved that BITC exhibits antitumor effects in bladder cancer by suppressing IGF1R, FGFR3, and mTOR, which is mediated by miR-99a expression. In this study, we identified the signal pathway involved in regulating miR-99a expression after BITC exposure in bladder cancer. Treatment with different BITC concentrations resulted in induction of miR-99a expression in bladder cancer cell lines. Activation of extracellular signal-regulated protein kinase (ERK) and c-jun N-terminal kinase was observed in bladder cancer after BITC treatment for 24 hours. Interestingly, by using a chemical inhibitor of candidate pathways, we found that only the ERK signal pathway is required for miR-99a expression. Furthermore, we evaluated the transcription factor that may contribute to miR-99a expression in response to BITC treatment. The results indicated that c-Jun/AP-1 was activated after BITC treatment. Moreover, we confirmed c-Jun/AP-1 activation through immunofluorescence and the luciferase reporter assay. The results showed that BITC treatment markedly improved nuclear translocation of c-Jun/AP-1 and luciferase activity dose dependently. Finally, pretreatment with the ERK inhibitor U0126 diminished c-Jun phosphorylation and transcriptional activation, suggesting that BITC elicits ERK/c-Jun signal transduction, which is responsible for miR-99a expression in bladder cancer. The present work identifies the mechanism involved in upregulation miR-99a after BITC treatment, which provides an explanation for BITC biological function in our previous work.