仇光宇、林致凡¹、林宜佳、蔡德甫、陳宏恩、黃一勝^2,3

新光吳火獅紀念醫院 外科部 泌尿科，¹新光吳火獅紀念醫院 中央實驗室；

²台北醫學大學 泌尿科；³天主教輔仁大學 醫學院 泌尿科

 

Kuang-Yu Chou , Ji-Fan Lin¹, Yi-Chia Lin, Te-Fu Tsai, Hung-En Chen, and Thomas I-Sheng Hwang^2,3

Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital¹, Department of Urology, Taipei Medical University², Division of Urology, School of Medicine, Fu-Jen Catholic University³, Taipei, Taiwan.

 

Purpose: The chemo-resistant was found in various types of cancer, and the autophagy induced by cisplatin treatment was considered to be one of the mechanisms. We previously demonstrated that forced expression of miR-30a-5p which targets autophagic genes ATG5 and beclin-1 (BECN1), and further enhanced cisplatin-induced apoptosis in human bladder cancer cells. Because of the precursor of miR-30a generates not only miR-30a-5p but also miR-30a-3p, we are interested in whether these two matured miR-30a (isomiRs) function simultaneously by targeting autophagic genes.

Materials and Methods: We established a cisplatin-resistant man bladder cancer cell line T24 (T24CR) by stepwise exposure of T24 cells to up to 40 μM of cisplatin. 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 these 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 T24 bladder cancer 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 cisplatin-treated or cisplatin resistant T24 and T24CR cells.

Conclusions: Our results demonstrate that miR-30a isomiRs can sensitize bladder cancer cells to cisplatin, via suppressing the expression of multiple autophagic genes. Increasing miR-30a levels in bladder cancer cell represents a novel strategy to enhance the efficacy of cisplatin chemotherapy.