Abstract
BackgroundCisplatin-based chemotherapy is recommended as the primary treatment for advanced bladder cancer (BC) with unresectable or metastatic disease. However, the benefits are limited due to the acquisition of drug resistance. The mechanisms of resistance remain unclear. Although there are some reports that some molecules are associated with cisplatin resistance in advanced BC, those reports have not been fully investigated. Therefore, we undertook a new search for cisplatin resistance-related genes targeted by tumor suppressive microRNAs as well as genes that were downregulated in cisplatin-resistant BC cells and clinical BC tissues.MethodsFirst, we established cisplatin-resistant BOY and T24 BC cell lines (CDDP-R-BOY, CDDP-R-T24). Then, Next Generation Sequence analysis was performed with parental and cisplatin-resistant cell lines to search for the microRNAs responsible for cisplatin resistance. We conducted gain-of-function analysis of microRNAs and their effects on cisplatin resistance, and we searched target genes comprehensively using Next Generation mRNA sequences.ResultsA total of 28 microRNAs were significantly downregulated in both CDDP-R-BOY and CDDP-R-T24. Among them, miR-486-5p, a tumor suppressor miRNA, was negatively correlated with the TNM classification of clinical BC samples in The Cancer Genome Atlas (TCGA) database. Transfection of miRNA-486-5p significantly inhibited cancer cell proliferation, migration, and invasion, and also improved the cells’ resistance to cisplatin. Among the genes targeted by miRNA-486-5p, we focused on enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase (EHHADH), which is involved in the degradation of fatty acids. EHHADH was directly regulated by miRNA-486-5p as determined by a dual-luciferase reporter assay. Loss-of-function study using EHHADH si-RNA showed significant inhibitions of cell proliferation, migration, invasion and the recovery of cisplatin sensitivity.ConclusionIdentification of EHHADH as a target of miRNA-486-5p provides novel insights into the potential mechanisms of cisplatin resistance in BC.
Highlights
Cisplatin-based chemotherapy is recommended as the primary treatment for advanced bladder cancer (BC) with unresectable or metastatic disease
Because miRNA-545-5p transfection did not suppress cell proliferation in CDDP-R BC cells and there was no correlations between miRNA-545-5p and the target gene (Supplementary Figure 2c), we focused on miRNA-486-5p as a strong candidate tumor suppressor that could overcome cisplatin resistance in this study
To determine whether EHHADH was directly regulated by miRNA-486-5p, we performed dual-luciferase reporter assays in CDDP-R BC cell lines
Summary
Cisplatin-based chemotherapy is recommended as the primary treatment for advanced bladder cancer (BC) with unresectable or metastatic disease. 50% of MIBC patients develop metastasis within 2 years, and the 5-year survival rate remains under 50% [1]. Advanced BC patients are generally treated with cisplatin-based combination chemotherapy as neoadjuvant and adjuvant therapy [2]. The overall survival rate (OS) has been extended by cisplatin, the median OS is only about 14 months after cisplatin based-chemotherapy [3, 4]. Because the molecular mechanisms of resistance to cisplatin in BC remain unclear, studies of the mechanism and novel prognostic markers to overcome cisplatin resistance are needed to improve outcomes in patients with BC. Knockdown of CTR1 reduces the intracellular accumulation of cisplatin and induces cisplatin resistance [8]. In spite of intense study of cisplatin resistance, it has not yet been overcome
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