表現微小核糖核酸30a標靶細胞自噬基因群增加抗順鉑(Cisplatin)抗藥性膀胱癌細胞之細胞凋亡
仇光宇1,4、林致凡2#、楊尚哲2、鄧雅旻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
Enhanced apoptosis in cisplatin-resistant human bladder cancer cells through forced expression of miR-30athat targets multiple autophagy related genes
Kuang-Yu Chou1,4, Ji-Fan Lin2, Shan-Che Yang2,Ya-Ming Teng2,Te-Fu Tsai 1,4, Hung-En Chen1, Yi-Chia Lin1,4, 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
Purpose:We previously demonstrated that forced expression of miR-30a-5p which targets ATG5 and beclin-1 (BECN1) enhanced cisplatin-induced apoptosis in human bladder cancer cells. Because the precursor of miR-30a generates not only miR-30a-5p but miR-30a-3p, we are interested in whetherthese two matured miR-30a function simultaneouslyby targeting autophagic genes.
Materials and Methods:We established a cisplatin-resistant 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 human BC cells. The expression level of miR-30a-3p amd mirR-30a-5p was detected by stem-loop miRNAqPCR. 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 expression level of miR-30a-3p or miR-30a-5p was elevated 6-8 fold at 48 hrs post-transfection in T24CR cells according to miRNAqPCR. The expression level of miR-30a targeting autophagic proteins was demonstrated to be down-regulated in both RNA and protein level. The autophagic levels in miRNA-transfected cells was decreased significantly compared to control. The apoptosis induction was increased in T24CR cells transfected with miR-30a-5p and miR-30a-3p, and further increased in cells transfected with both miRNAs.
Conclusions:Our results demonstrate that miR-30a-3p and miR-30a-5p generated from a single precursor transcript (miR-30a) can target autophagic genes and overcome the cisplatin-resistant in human bladder cancer cells.This finding could extent to other drug-resistant model by targeting induced autophagy.
Running title: miR-30a inhibits autophagy and enhanced apoptosis in cisplatin-resistant bladder cancer cells.