miR-30a標靶多個細胞自噬相關基因並增強順鉑(Cisplatin)
在膀胱癌細胞之抗癌效果
仇光宇1 、林致凡2、楊尚哲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
miR-30a isomiRs targets multiple autophagy related genes and sensitizes human bladder cancer cells to cisplatin treatment
Kuang-Yu Chou1, Ji-Fan Lin2, Shan-Che Yang2, 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.
Purpose: Autophagy has been considered as a chemo-resistant mechanism in various types of cancer, including bladder cancer (BC). We previously demonstrated that forced expression of miR-30a-5p which targets ATG5 and beclin-1 (BECN1) enhanced cisplatin-induced apoptosis in human BC cells. Because the precursor of miR-30a generates not only miR-30a-5p but miR-30a-3p, we are interested in whether these two matured miR-30a (isomiRs) function simultaneously by targeting multiple autophagy-related genes.
Materials and Methods: To elevate the expression level of miR-30a-3p and miR-30a-5p, a small RNA expression vector bearing matured sequence of miR-30a-3p (pSM-30a-3p) or miR-30a-5p (pSM-30a-5p) was constructed and transfected into BC cells. The expression level of miR-30a-3p and mirR-30a-5p was detected by stem-loop miRNA qPCR. Protein level of predicted targeting genes that involved in autophagy formation including ATG3, ATG4C, ATG5, ATG7, ATG12 and BECN1, was accessed by QPCR and Western blot. Autophagy detection in cisplatin-treated cells was performed by monitoring LC3-II processing by Western blot. Induction of apoptosis in cisplatin-resistant cells with or without the over-expressed miR-30a-3p or miR-30a-5p was detected by the detection of cleaved caspase-3 and PARP.
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 and ATG2B, ATG3, ATG4C, ATG7, ATG12, ATG14, ATG16L1, and BECN1 were predicted as targets for miR-30a-3p by TargetScan. Forced expression of miR-30a-5p or miR-30a-3p significantly reduced the expression level of ATG5 and BECN1 or ATG7, ATG12, and BECN1, respectively. Co-transfection of these miR-30a isomiRs significantly reduced LC3-II processing and enhanced apoptosis in T24 cells with or without cisplatin treatment.
Conclusions: Our results demonstrate that miR-30a-3p and miR-30a-5p generated from a single precursor transcript (miR-30a) can target autophagic genes. Inhibition of basal or cisplatin-induced autophagy enhances apoptotic cell death. These findings could extent to other drug-resistant model by targeting induced autophagy.