MiR-193a-5p Targets the Coding Region of AP-2α mRNA and Induces Cisplatin Resistance in Bladder Cancers

Abstract

Transcription factor AP-2 alpha (AP-2α or TFAP2A) is a newly identified prognostic marker of chemotherapy; its expression is positively correlated with chemosensitivity and survival of cancer patients. Using computational programs, we predicted that the coding region of AP-2α gene contains a potential miRNA response element (MRE) of miR-193a-5p, and the single nucleotide polymorphism (SNP) site (c.497A>G, rs111681798) resides within the predicted MRE. The results of luciferase assays and Western blot analysis demonstrated that miR-193a-5p negatively regulated the expression of AP-2α proteins, but have no influence on the mutant AP-2α (c.497A>G). Infection with lentiviral AP-2α gene or miR-193a-5p inhibitor in the bladder cancer cells decreased migration and cisplatin resistance, while knockdown of AP-2α gene or overexpression of miR-193a-5p in the urothelial cell line SV-HUC-1 increased migration and cisplatin resistances. We concluded that miR-193a-5p induced cisplatin resistance by repressing AP-2α expression in bladder cancer cells.

Keywords: AP-2α; bladder cancer; cisplatin resistance; miR-193a-5p; single nucleotide polymorphism..

Figure 1

The miR-193a-5p targets the coding region (CDS) of AP-2α mRNA. (A) Diagram of the binding between miR-193a-5p seed sequence and the AP-2α CDS. (B) Myc-tagged wild-type or mutant AP-2α expression plasmids were co-transfected with miR-193a-5p mimics into 293T cells. At 24 h post-transfection, cell lysates were prepared and subjected to Western blotting assay using anti-Myc antibody. (C) Each luciferase construct was co-transfected with miR-193a-5p mimics into 293T cells. At 24 h post-transfection, the luciferase activity was examined. The firefly luciferase activity was normalized to Renilla luciferase activity. Data are shown as the mean+SD of three independent experiments. *, p<0.05; **, p<0.01.

Figure 2

Influence of miR-193a-5p on AP-2α expression. (A)Quantitative analysis of miRNA-193a-5p expression on various cell lines by realtime PCR. (B) Expression of AP-2α protein in three cell lines using Western blotting. (C)(D) Expression of AP-2α protein in UM-UC-3 cells (C) and SV-HUC-1 cells (D). (E) Influence of miR-193a-5p on AP-2α downstream targets. All the cells were infected with lentivirus, and harvested for Western blot at 72 h after infection.

Figure 3

Representative pictures of wound healing dynamics. UM-UC-3 (A, C) and SV-HUC-1 cells (B, D) infected with the indicated lentivirus were cultured on a 24-well plate until 100% confluence and then wounded with a 100 μL pipette tip. Migration photos were captured at 0, 24, and 48h hours after scratching.

Figure 4

Influence of miR-193a-5p and AP-2α on cisplatin sensitivity. UM-UC-3 cells were infected with lentiviral miR-193a-5p inhibitor or AP-2α gene. At 72h after transduction, cells were seeded on 96-well plates and treated with different concentration of cisplatin for 36 h. The cell viability was determined by MTT assay.