異硫氰酸苄酯(BITC)調控微小核糖核酸-99a在膀胱癌細胞中的雙重角色:誘發細胞自嗜和細胞凋亡
蔡德甫1,4、林致凡2、林宜佳1,4、陳宏恩1、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
Dual roles of benzyl isothiocycante-induced miR-99a in bladder cancer cells: Inducing autophagy and apoptosis
Te-Fu Tsai1,4, Ji-Fan Lin2,Yi-Chia Lin1,4,Hung-En Chen1, Kuang-Yu Chou1,4, 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, Division of Urology, 4School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan.
Purpose: Bladder cancer (BC) with increasing incidence is a common cancer worldwide. The anti-tumor properties of benzyl isothiocyanate (BITC) has been demonstrated in various types of cancer including BC. MicroRNAs (miRNAs) are small (~22 nt) non-coding RNAs that are frequently dysregulated in cancer, and have shown to play important roles in tumorigenesis. BITC induces the expression of miR-99a-5p which is reported to down-regulated in BC. We found that protective autophagy was induced in BC by BITC through activation of miR-99a-5p, which targets mTOR. However, certain pro-survival proteins such as insulin-growth factor 1 type I receptor (IGF1R) and fibroblast growth factor type III receptor (FGFR3) are also reported to be targeted by miR-99a-5p. In this study, we aim to find out the role of BITC-induced miR-99a-5p regulating autophagy and apoptosis using BC cells.
Materials and Methods: Human BC cell lines (5637 and T24) and immortalized urothelial cells (SV-Huc) were used in this study. Detection of miR-99a-5p expression level in BITC-treated cells was performed by miRNA qPCR. We utilized a luciferase reporter vector bearing anti-sense miR-99a-5psequence to confirmthe up-regulation of miR-99a-5p and act as an inhibitor to the miR-99a-5p in BITC-treated BC cells. To facilitate the overexpression of miR-99a-5p, small RNA expression vector (pSM) was constructed. Expression levels of IGF1R, FGFR3, mTOR, LC3-II protein was detected by Western blot.
Results: BITC induced the expression of miR-99a-5p in BC cells was detected by miRNA qPCR. The results showed that miR-99a-5p which is down regulated in BC cells was up-regulated by BITC treatment.Treatment of BITC resulted in the decreased expression of IGF1R, FGFR3, mTOR; and increased expression of LC3-II, suggesting the induction of autophagy and apoptosis in the same time. In the other hand, overexpression of miR-99a-5p mimic the effects of BITC on the expression of these proteins. And the expression of these proteins was restored, if not all, in cells transfected with antisense miR-99a-5p prior to the treatment of BITC, suggesting that BITC regulated the expression of IGF1R, FGFR3, mTOR and LC3-II through miR-99a-5p.
Conclusions: Our results provided the first clue that miR-99a-5p that regulated by BITC induces autophagy and apoptosis through targeting multiple proteins. Understand the action mechanism of miR-99a-5p which is reported to be dysregulated in various cancers may provide further direction for novel treatment against cancer.