異硫氰酸苄酯(BITC)調控微小核糖核酸-99a抑制mTOR表現
並在膀胱癌細胞中誘發細胞自嗜
蔡德甫1,4、林致凡2、楊尚哲2、陳宏恩1、林宜佳1,4、仇光宇1,4、黃一勝1,3,4
1新光醫院外科部泌尿科、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 up-regulates miR-99a-5p and induces autophagy by suppressing mTOR expression in human bladder cancer cells
Te-Fu Tsai1,4, Ji-Fan Lin2, Shan-Che Yang2, Hung-En Chen1, Yi-Chia Lin1,4, Kuang-Yu Chou1,4, and Thomas I-Sheng Hwang1,3,4
1Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital, 2Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital, 3Department of Urology, Taipei Medical University., 4Division of Urology, School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan.
Purpose: The development of novel methods for BC therapy, particularly for recurring BC, is warranted. Benzyl isothocyanate (BITC) is a natural compound that produced by Brassicaceae species, and displays anticarcinogenic activities. Besides apoptosis induction, BITC also induces autophagy through inhibiting mTOR. Human miR-99a-5p was reported to be down-regulated in BC and it potentially targets to mTOR. We therefore hypothesized that BITC may induce autophagy through regulating miR-99a-5p to suppress mTOR expression and its downstream singaling.
Materials and Methods: Human BC cell lines (5637 and T24) were used in this study. To facilitate the overexpression of miR-99a-5p, the matured miR-99a-5p was inserted to a small RNA expression vector (pSM) to generate pSM-99a.Detection of miR-99a-5p expression level in pSM-99a transfected or BITC-treated cells was performed by miRNA qPCR. We utilized a luciferase reporter vector bearing anti-sense miR-99a-5psequences to confirm the up-regulation of miR-99a-5p, and to act as an inhibitor to the miR-99a-5p in BITC-treated BC cells. Protein expression level of LC3-II, mTOR, IGF1R, FGFR3 was monitored in pSM-99a transfected or BITC-treated cells using Western blot.
Results: We first verified that BITC decreased cell viability by induction of apoptosis in human BC cells, as it does in prostate cancer cells. BITC also induced autophagy through decreased the mTOR expression level. Over-expression of miR-99a-5p, which is down-regulated in bladder cancer cells, resulted in decreased cell viability and inhibition of mTOR protein level. We found the expression level of mTOR, IGF1R and FGFR3 that are direct targets of miR-99a-5p was decreased in BITC-treated as well as the pSM-99a transfected BC cells. These results lead us to detect the expression level of miR-99a-5p in BITC-treated cells. The expression level of miR-99a-5p in BITC-treated BC cells was detected by two methods: (a) miRNA qPCR and (b) luciferase reporter assays. The results showed that miR-99a-5p was up-regulated by BITC treatment. BITC induces autophagy by inhibiting mTOR expression; and forced expression of miR-99a-5p was able to mimic the autophagy induction and mTOR inhibition asin BITC-treated cells. The transfection of luciferase reporter that competes and inhibits BITC-induced miR-99a-5p resulted in the restoration of mTOR expression and decreased level of autophagy, suggesting autophagy induction in BITC-treated cells is partially, if not all, through the up-regulation of miR-99a-5p.
Conclusions: Our results indicated that miR-99a-5p plays animportant role in BITC-induced autophagy in BC cells.