Long non-coding RNA GAS5 Modulates Gemcitabine Efficacy in Bladder Cancer via Regulation of Deoxycytidine Kinase Expression
Gemcitabine is a common used agent for therapy in bladder cancer. Our objective was to characterize the ability of Long non-coding RNA GAS5 to modulate the gemcitabine efficacy.
Expression of GAS5 was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR) in 30 patients following radical cystectomy and gemcitabine treatment. All subjects signed written informed consent. The study was approved by the Hospital Ethics Boards prior to initiation. The correlation between GAS5 and deoxycytidine kinase (DCK) was further examined by evaluating DCK expression in cells that either overexpressed or knocked down GAS5. Gemcitabine efficacy was identified by CCK-8 assays. DCK and HuR were analyzed by western blotting and immunofluorescence.
GAS5 was significantly downregulated in bladder cancer tissues compared with the paired adjacent non-tumorous tissues. Lower GAS5 expression levels were associated with advanced pathological stages and poor overall survival (Fig. 1). LncRNA GAS5 expression is also lower in T24, 5637 and SW780 compared with the human normal urothelial cell line (Sv-Huc-1). GAS5 level was well correlated with the sensitivity of the cancer cells toward gemcitabine. Gemcitabine can dose-dependently increase GAS5 level in bladder cancer cells. Of note, pretreated gemcitabine attenuated GAS5-induced with a second dose of gemcitabine. GAS5-overexpressing cells displayed an elevated level of cell death induced by gemcitabine. Accordingly, knockdown of GAS5 in bladder cancer cells by small interfering RNA conferred tolerance to gemcitabine-induced cytotoxicity (Figure 3C, 3D). DCK is the rate-limiting enzyme responsible for conversion of gemcitabine to active antimetabolite. By knocking down GAS5 in bladder cancer cells, we experimentally confirmed that GAS5 regulates DCK expression. Furthermore, we revealed that proapoptotic function of GAS5 was correlated with promoting HuR nuclear export and the biological consequence of deoxycytidine kinase expression (Fig. 2).
This study shows that long non-coding RNA GAS5 modulates HuR-mediated DCK expression, and GAS5 low expression confers gemcitabine tolerance. The expression of GAS5 may represent a novel companion diagnostic and target to undermine chemotherapy resistance.
Funding: This work was supported by grants from the National Natural Science Foundation of China (81502203), the Natural Science Foundation of Jiangsu Province (BK20150097)