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.
Conclusion: 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.