微小RNA 30a-5p 過度表現抑制順鉑引起的細胞自噬
仇光宇1,3、林致凡4、林宜佳1,3、蔡德甫1,3、陳宏恩1、黃一勝1,2,3
1新光醫院 外科部 泌尿科; 2台北醫學大學醫學系 泌尿學科; 3輔仁大學醫學系 泌尿學科; 4新光醫院中央實驗室
Inhibition of cisplatin-induced autophagy by overexpression of miR-30a-5p
Kuang-Yu Chou1,3, Ji-Fan Lin4,Yi-Chia Lin1,3,Te-Fu Tsai1,3, Hung-En Chen1, and Thomas I-Sheng Hwang1,2,3
1Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital, Department of Urology,
2Taipei Medical University, Division of Urology, 3School of Medicine, Fu-Jen Catholic University, 4Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
Purpose:We previously show that autophagy is activated and is contributed to cisplatin-resistance in cisplatin-treated bladder cancer (BC) cells. miR-30a-5p was down-regulated in BC and potentially targets to ATG5 and beclin-1 (BECN1, both are involved in autophagy progression. In this study, we overexpressed miR-30a-5p to inhibits autophagy induced by cisplatin treatment.
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.Autophagy detection in cisplatin-treated cells was performed by detecting LC3-II processing by Western blot. Overexpression of miR-30a-5p was achieved by transfecting small RNA expression vector bearing matured sequence of miR-30a-5p (pSM-30a). The expression level of miR-30a-5p was detected by stem-loop miRNA qPCR. Protein level of ATG5 and beclin-1 (BECN1) was accessed by Western blot. The cell viability was measured using WST-1 reagent. Induction of apoptosis in cisplatin-treated cells with or without the over-expressed miR-30a-5p was detected by the activation of caspase 3/7 activity.
Results:The autophagy activity in BC cells increased after cisplatin treatment as indicated by the enhanced processing of LC3-II.ATG5 and BECN1 were predicted as 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 and that 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.