miR-214-3p Regulates Multi-Drug Resistance and Apoptosis in Retinoblastoma Cells by Targeting ABCB1 and XIAP

Abstract

Background: MicroRNAs (miRNAs) have been shown to contribute to the initiation and progression of human cancer, including retinoblastoma. However, expression levels and potential roles of miRNAs in retinoblastoma remain largely unknown. In this study, we aimed to identify dysregulated miRNAs and explore their functional roles in the development of retinoblastoma.

Material and methods: First, miRNA expression profiling in retinoblastoma tissues was performed via microarray analysis. To evaluate the involvement of miR-214-3p in multi-drug resistance, gain-of-function experiments were employed in vitro and in vivo. Bioinformatics analysis, luciferase reporter assay, qRT-PCR and Western blot were used to investigate the underlying mechanisms.

Results: Here, we identified 57 up-regulated and 34 down-regulated miRNAs. Among them, miR-214-3p was the most significantly decreased. We found that miR-214-3p level was positively correlated with clinical outcome and chemotherapy response. Overexpression of miR-214-3p significantly sensitized retinoblastoma cells to multiple chemodrugs and promoted cell apoptosis in vitro and in vivo. Further investigations revealed that miR-214-3p directly regulated ABCB1 and XIAP expression through interacting with the 3' untranslated regions (3'UTRs). Pearson correlation analysis showed that miR-214-3p expression in retinoblastoma tissues was negatively correlated with ABCB1 and XIAP expression. We also observed that overexpression of ABCB1 or XIAP partly reversed the chemoresistance inhibition-induced by miR-214-3p overexpression.

Conclusion: Our data demonstrate that miR-214-3p functions as a tumor suppressor to inhibit the chemoresistance in retinoblastoma, suggesting that miR-214-3p might be potential diagnostic and therapeutic targets for retinoblastoma treatment.

Keywords: ABCB1; XIAP; chemoresistance; miR-214-3p; retinoblastoma.

Figure 1

miR-214-3p is down-regulated in retinoblastoma tissues and cell lines. (A) Heatmap of the top 50 differentially expressed miRNAs between 3 retinoblastoma tissues and 3 normal retina tissues (fold change >2 and P < 0.05) determined using miRNA array analysis. (B) The miR-214-3p expression level in 56 retinoblastoma tissues quantified by qRT-PCR analysis. Expression levels were shown as log2-fold change to age-matched normal retina tissues. (C) The relative expression level of miR-214-3p in 56 retinoblastoma tissues and 15 age-matched normal retina tissues. (D) The relative expression level of miR-214-3p in human retinoblastoma cell lines WERI-RB1, SO-RB50, Y79, and human retinal pigment epithelial cell line ARPE-19. (E) Kaplan–Meier analysis of overall survival in 56 retinoblastoma patients was analyzed using Log rank test according to miR-214-3p expression compared with normal retina tissues. Data indicate mean ± SD of at least three independent experiments; Student’s t-test. **P<0.01.

Figure 2

miR-214-3p enhances the sensitivity to multi-drug in vitro. (A) Resistance index of VCR, CBP, DDP, VP-16 and 5-Fu in SO-RB50/VCR and SO-RB50/CBP cells compared with their parallel cells SO-RB50. (B) The miR-214-3p expression level in SO-RB50/VCR, SO-RB50/CBP cells and their parallel cells SO-RB50. (C) The miR-214-3p expression level in SO-RB50/VCR and SO-RB50/CBP cells transfected with miR-214-3p mimics and miRNA negative control. (D) and (E) Resistance index of VCR, CBP, DDP, VP-16 and 5-Fu in SO-RB50/VCR (D) and SO-RB50/CBP (E) cells transfected with miR-214-3p mimics and miRNA negative control. (F) Flow cytometry assays were performed to analyze the effect of miR-214-3p overexpression on cell apoptosis in SO-RB50/VCR and SO-RB50/CBP cells treated with VCR or CBP. Data indicate mean ± SD of at least three independent experiments; Student’s t-test. *P<0.05, **P<0.01.

Figure 3

miR-214-3p enhances the sensitivity to multi-drugs in vivo. SO-RB50 cells that stably overexpressed miR-214-3p (pSuper-miR-214-3p) or pSuper were subcutaneously injected into nude mice treated with VCR (1.0 mg/kg) or CBP (50 mg/kg) twice weekly. After 4 weeks treatment, the mice were sacrificed and the tumors were isolated and weighed. (A) Representative images. (B) and (D) Tumor volume of mice treated with VCR (B) or CBP (D). (C) and (E) Tumor weight mice treated with VCR (C) or CBP (E). Data indicate mean ± SD of at least three independent experiments; Student’s t-test. **P<0.01.

Figure 4

ABCB1 and XIAP are direct target genes of miR-214-3p. (A) Predicted binding sites of miR-214-3p in the 3’UTR of ABCB1 and XIAP analyzed using TargetScan 7.2. (B) Protein expression level of ABCB1 and XIAP in SO-RB50/VCR and SO-RB50/CBP cells transfected with miR-214-3p mimics or miRNA negative control. (C) and (D) Luciferase assays were performed after HEK-293T cells were co-transfected wt or mut 3’UTR of ABCB1 (C) and XIAP (D) with miR-214-3p mimics or miRNA negative control for 48 hrs. (E) and (F) The relative mRNA expression level of ABCB1 (E) and XIAP (F) in 56 retinoblastoma tissues and 15 age-matched normal retina tissues. (G) and (H) The correlation between miR-214-3p and ABCB1 (G) or XIAP (H) in 56 retinoblastoma tissues. Data indicate mean ± SD of at least three independent experiments; Student’s t-test. **P<0.01.

Figure 5

ABCB1 and XIAP rescue miR-214-3p-suppressed chemoresistance. (A) Protein expression level of ABCB1 in SO-RB50/VCR and SO-RB50/CBP cells transfected with miR-214-3p mimics or miRNA negative control and pcDNA-ABCB1 or pcDNA 3.1. (B) Protein expression level of XIAP in SO-RB50/VCR and SO-RB50/CBP cells transfected with miR-214-3p mimics or miRNA negative control and pcDNA-XIAP or pcDNA 3.1. (C) and (D) Resistance index of VCR, CBP, DDP, VP-16 and 5-Fu in SO-RB50/VCR (C) and SO-RB50/CBP (D) cells transfected with miR-214-3p mimics or miRNA negative control and pcDNA-ABCB1 or pcDNA-XIAP. (E) and (F) Flow cytometry assays were performed to analyze the apoptosis rate of SO-RB50/VCR (E) and SO-RB50/CBP (F) cells transfected with miR-214-3p mimics or miRNA negative control and pcDNA-ABCB1 or pcDNA-XIAP treated with VCR or CBP. Data indicate mean ± SD of at least three independent experiments; Student’s t-test. *P<0.05, **P<0.01.