MicroRNA-93-5p increases multidrug resistance in human colorectal carcinoma cells by downregulating cyclin dependent kinase inhibitor 1A gene expression

Abstract

Multidrug resistance (MDR) impedes successful chemotherapy in colorectal carcinoma (CRC) and emerging evidence suggests that microRNAs (miRs) are involved in the development of MDR. In the present study, the role of miR-93-5p in the modulation of drug resistance in CRC was investigated using HCT-8 and MDR HCT-8/vincristine (VCR) cell lines. The results demonstrated upregulated expression of miR-93-5p and MDR protein 1 (MDR1) in HCT-8/VCR cells, compared with the parental HCT-8 cells. Furthermore, cyclin-dependent kinase inhibitor 1A (CDKN1A) was identified as a potential target of miR-93-5p using miR target analysis tools, including PicTar, TargetScan and miRanda. In addition, inhibition of miR-93-5p expression in HCT-8/VCR cells markedly downregulated MDR1 gene expression, upregulated CDKN1A gene expression and induced cell cycle arrest in G₁. Conversely, the overexpression of miR-93-5p in HCT-8/VCR cells upregulated MDR1 gene expression, downregulated CDKN1A gene expression and promoted G₁/S transition. Furthermore, the in vitro drug sensitivity assay performed suggested that downregulation of miR-93-5p enhanced the sensitivity of HCT-8/VCR cells to VCR, while the upregulation of miR-93-5p decreased the sensitivity of HCT-8 cells to VCR. In conclusion, the results of the present study suggest that miR-93-5p serves a role in the development of MDR through downregulating CDKN1A gene expression in CRC.

Keywords: colorectal carcinoma; cyclin dependent kinase inhibitor 1A; microRNA-93; multidrug resistance; multidrug resistance protein 1.

Figure 1.

(A) Relative expression levels of miR-93-5p in HCT-8 and HCT-8/VCR cells, detected using RT-qPCR. Relative expression of miR-93-5p was normalized to U6 small nuclear RNA. (B) Relative expression levels of MDR1 mRNA in HCT-8 and HCT-8/VCR cells, as measured using RT-qPCR. GAPDH was used as an internal loading control. Values are presented as the mean ± standard deviation from three independent experiments. **P<0.01 vs. HCT-8 cells. (C) MDR1 protein levels were measured using western blot analysis. β-actin was used as an internal loading control. (D) MDR1 protein expression was quantified through image analysis. Values are presented as the mean ± standard deviation from three independent experiments. miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; MDR1, multidrug resistance protein 1; VCR, vincristine.

Figure 2.

(A) Relative expression levels of MDR1 mRNA in HCT-8/VCR cells transfected with miR-93-5p mimic, miR-93-5p inhibitor or a NC were measured using the reverse transcription-quantitative polymerase chain reaction 48 h following the transfection. GAPDH was used as an internal loading control. (B) MDR1 protein levels in HCT-8/VCR cells transfected with miR-93-5p mimic, miR-93-5p inhibitor or a NC were analyzed using western blot analysis at 72 h following transfection. β-actin was used as an internal loading control. (C) MDR1 protein expression was quantified through image analysis. Values are presented as the mean ± standard deviation from three independent experiments. **P<0.01 vs. the NC group. miR, microRNA; MDR1, multidrug resistance protein 1; NC, negative control; VCR, vincristine.

Figure 3.

miR-93-5p promotes efflux of Rh-123 in HCT-8/VCR cells. (A) The release index of Rh-123 in HCT-8/VCR cells transfected with miR-93-5p mimic, miR-93-5p inhibitor or a NC was determined following 1 h incubation with 20 µM Rh-123. The fluorescence intensity of Rh-123 in cells was measured using fluorescence activated cell sorting. (B) Quantification of Rh-123 fluorescence intensity. Values are presented as the mean ± standard deviation from three independent experiments. *P<0.05; **P<0.01 vs. the NC group. (C) A total of 24 h following transfection of HCT-8 and HCT-8/VCR cells with miR-93-5p mimic, miR-93-5p inhibitor or a NC, cells were treated with increasing concentrations of VCR for 72 h. Inhibition of cell viability was subsequently measured using the Cell Counting kit-8 assay. miR, microRNA; Rh-123, rhodamine 123; NC, negative control; MDR1, multidrug resistance protein 1; VCR, vincristine; FL1-H, fluorescence 1 height.

Figure 4.

(A) Relative expression levels of CDKN1A mRNA in HCT-8/VCR cells transfected with miR-93-5p mimic, miR-93-5p inhibitor or an NC were determined using the reverse transcription-quantitative polymerase chain reaction 48 h following transfection. GAPDH was used as an internal loading control. (B) CDKN1A protein levels in HCT-8/VCR cells transfected with miR-93-5p mimic, miR-93-5p inhibitor or an NC were analyzed using western blotting 72 h following transfection. β-actin was used as an internal control. (C) Relative expression of CDKN1A protein was quantified through image analysis. Values are presented as the mean ± standard deviation from three independent experiments. *P<0.05; **P<0.01 vs. the NC group. miR, microRNA; CDKN1A, cyclin-dependent kinase inhibitor 1A; NC, negative control; VCR, vincristine.

Figure 5.

(A) Cell cycle distribution of HCT-8/VCR cells transfected with miR-93-5p mimic, miR-93-5p inhibitor or an NC was analyzed 72 h following transfection using flow cytometry. Overexpression of miR-93-5p induced cell cycle progression in HCT-8/VCR cells. (B) Histogram showing the percentage of cells in G₀/G₁, S and G₂/M cell cycle phases. Values are presented as the mean ± standard deviation from three independent experiments. miR, microRNA; NC, negative control; FL2-A, FL2-area; VCR, vincristine.