HIF-1α-dependent miR-424 induction confers cisplatin resistance on bladder cancer cells through down-regulation of pro-apoptotic UNC5B and SIRT4.

Background: Chemo-resistance of bladder cancer has been considered to be one of the serious issues to be solved. In this study, we revealed pivotal role of miR-424 in the regulation of CDDP sensitivity of bladder cancer cells.

Methods: The cytotoxicity of cisplatin and effect of miR-424 were assessed by flow cytometry and TUNEL. Transcriptional regulation of miR-424 by HIF-1α was assessed by Chromatin immunoprecipitation (ChIP). Effect of miR-424 on expression of UNC5B, SIRT4 (Sirtuin4) and apoptotic markers was measured by QRT-PCR and/or Western blot. The regulation of miR-424 for UNC5B and SIRT4 were tested by luciferase reporter assay. The 5637-inoculated nude mice xenograft model was used for the in vivo study. The clinical significance of miR-424 was demonstrated mainly through data mining and statistical analysis of TCGA.

Results: In this study, we have found for the first time that cisplatin (CDDP) induces the expression of miR-424 in a HIF-1α-dependent manner under normoxia, and miR-424 plays a vital role in the regulation of CDDP resistance of bladder cancer cells in vitro. Mechanistically, we have found that UNC5B and SIRT4 are the direct downstream target genes of miR-424. CDDP-mediated suppression of xenograft bladder tumor growth was prohibited by the addition of miR-424, whereas ectopic expression of UNC5B or SIRT4 partially restored miR-424-dependent decrease in CDDP sensitivity of bladder cancer 5637 and T24 cells. Moreover, knockdown of UNC5B or SIRT4 prohibited CDDP-mediated proteolytic cleavage of PARP and also decreased CDDP sensitivity of these cells. Consistently, the higher expression levels of miR-424 were closely associated with the poor clinical outcome of the bladder cancer patients. There existed a clear inverse relationship between the expression levels of miR-424 and pro-apoptotic UNC5B or SIRT4 in bladder cancer tissues.

Conclusions: Collectively, our current results strongly suggest that miR-424 tightly participates in the acquisition/maintenance of CDDP-resistant phenotype of bladder cancer cells through down-regulation of its targets UNC5B and SIRT4, and thus combination chemotherapy of CDDP plus HIF-1α/miR-424 inhibition might have a significant impact on hypoxic as well as normoxic bladder cancer cells.