表現微小核糖核酸30a-5p標靶ATG5與Beclin-1增加順鉑(cisplatin)在人類膀胱癌細胞中之抗癌效果
仇光宇1 #、林致凡2#、蔡德甫1、陳宏恩1、林宜佳1、黃一勝1,2,3,4
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
Forced expression of miR-30a-5p sensitizes bladder cancer cells to cisplatin
via targeting ATG5 and Beclin-1
Kuang-Yu Chou1#, Ji-Fan Lin2#, Te-Fu Tsai 1, Hung-En Chen1, Yi-Chia Lin1 , 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.
# These authors contributed equally to this work.
Purpose: Autophagy is activated and may contributed to cisplatin-resistance in cisplatin-treated bladder cancer (BC) cells. It is reasonable to speculate that Inhibition of autophagy enhances the anti-cancer effects of cisplatin in BC cells. In this study, we characterized the role of miR-30a-5p, which is down-regulated in BC cells, in the coordination of apoptosis and autophagy by accessing its potential targeting protein, ATG5 and beclin-1 (BECN1).
Materials and Methods: The BC cell lines, 5637 (grade II) and T24 (grade III) and immortalized human uroepithelium cells (SV-HUC-1) were used in this study. To elevate the expression level of miR-30a-5p, a small RNA expression vector bearing matured sequence of miR-30a-5p (pSM-30a) was constructed and transfected into human BC cells. The expression level of miR-30a-5p was detected by stem-loop miRNA qPCR. Protein level of ATG5 and BECN1, both are predicted targets of miR-30a-5p, was accessed by Western blot. Autophagy detection in cisplatin-treated cells was performed by monitoring LC3-II processing by Western blot. Induction of apoptosis in cisplatin-treated cells with or without the over-expressed miR-30a-5p was detected by the detection of cleaved caspase-3 and PARP.
Results: The expression level of miR-30a-5p was elevated up to 8 fold in pSM-30a transfected BC cells according to miRNA qPCR. The autophagy activity in BC cells increased after cisplatin treatment as indicated by the enhanced processing of LC3-II. As ATG5 and BECN1 were predicted targets for miR-30a-5p by TargetScan, forced expression of miR-30a-5p significantly reduced the expression level of ATG5, BECN1 and LC3-II induced by cisplatin. The blockage of autophagy by miR-30a-5p expression or bafilomycin A1 (Baf A1) significantly decreased cell viability and increased apoptosis in cisplatin-treated BC cells.
Conclusions: Our results demonstrate that miR-30a-5p can sensitize BC cells to cisplatin via suppressing ATG5 and BECN1 expression, therefore, increasing miR-30a-5p level in BC represents a novel strategy to enhance the efficacy of cisplatin therapy during cancer treatment.
Running title: miR-30a-5p enhances cisplatin-induced apoptosis via targeting autophagy.
Keywords: Autophagy, apoptosis, BECN1, bladder cancer, miR-30a-5p.