MiR-424-3p suppresses galectin-3 expression and sensitizes ovarian cancer cells to cisplatin

Abstract

Purpose: Assessment of miR-424-3p mimic capability to sensitize SK-OV-3 and TOV-21G ovarian cancer cells to cisplatin by decreasing the expression of galectin-3, which is an anti-apoptotic protein overexpressed in ovarian cancer and associated with resistance to chemotherapy.

Methods: We performed a reverse transfection of miR-424-3p mimic into SK-OV-3 and TOV-21G ovarian cancer cells, followed by Real Time™ RT-PCR analysis of the expression of miR-424-3p and galectin-3 mRNA as well as ELISA assay for galectin-3 protein level. Next, we studied the viability (XTT assay), proliferation (EdU incorporation assay), and apoptosis (ELISA assay) of the both cell lines transfected with the mimic and treated with cisplatin.

Results: We demonstrated that miR-424-3p mimic effectively transfects into SK-OV-3 and TOV-21G ovarian cancer cells in which it significantly suppresses the expression of galectin-3 at the protein level, but not at the mRNA level. Reverse transfection of both cell lines with the mimic, followed by treatment with cisplatin, resulted in a reduction in cell viability and proliferation as well as an increase in the induction of apoptosis.

Conclusions: MiR-424-3p mimic sensitizes SK-OV-3 and TOV-21G ovarian cancer cells to cisplatin by decreasing the expression of galectin-3.

Keywords: Cisplatin; Drug resistance; Galectin-3; Ovarian cancer; miR-424-3p.

Fig. 1

Human ovarian cancer cells reverse transfected with 5′-FAM-labeled miR-424-3p mimic (a SK-OV-3; b TOV-21G) or the negative control (c SK-OV-3; d TOV-21G). Images were captured at ×100 magnification by an Eclipse Ti (Nikon Instruments Inc.) fluorescence microscope. a and b Presence of green fluorescence indicated successfully transfected cells (exemplary transfected cells are marked with white arrows and untransfected cells with black arrows). The efficiency of reverse transfection (the ratio of cells successfully transfected with 5′-FAM-labeled miR-424-3p mimic to the total number of cells) was approximately 97% in both cell lines

Fig. 2

Expression of miR-424-3p in SK-OV-3 (a) and TOV-21G (b) human ovarian cancer cells at 24 h, 48 h, and 72 h after the reverse transfection of miR-424-3p mimic into both cell lines. The expression was analyzed by the Real Time™ stem-loop RT-PCR technique ($2^{-\mathrm{\Delta }\mathrm{\Delta }{C}_{\text{q}}}$ method). The data are shown as mean ± SD of triplicate experiments. A two-way analysis of variance (ANOVA), followed by post hoc Tukey’s HSD test, was used to test statistical differences between the studied groups. p values less than 0.05 were considered as significant. All bars on the charts, which are marked with the same letter (even if it is only one common letter among many), are not significantly different

Fig. 3

Expression of galectin-3 at the mRNA and protein levels in SK-OV-3 (a mRNA; c protein) and TOV-21G (b mRNA; d protein) human ovarian cancer cells at 24 h, 48 h, and 72 h after the reverse transfection. The expression was analyzed by Real Time™ RT-PCR technique at the mRNA level ($2^{-\mathrm{\Delta }\mathrm{\Delta }{C}_{\text{q}}}$ method) and by ELISA assay at the protein level. The data are shown as mean ± SD of triplicate experiments. A two-way analysis of variance (ANOVA), followed by post hoc Tukey’s HSD test, were used to test statistical differences between the studied groups. A p values less than 0.05 were considered as significant. All bars on the charts, which are marked with the same letter (even if it is only one common letter among many), are not significantly different

Fig. 4

Analysis of human ovarian cancer cells: viability by XTT assay (a SK-OV-3; b TOV-21G); proliferation by EdU incorporation assay (c SK-OV-3; d TOV-21G); apoptosis by quantitative determination of cytoplasmic histone-associated-DNA-fragments in ELISA assay, followed by the calculation of enrichment factor for each studied group (e SK-OV-3; f TOV-21G). The cells were reverse transfected and treated with cisplatin for 24 h, at 48 h after the reverse transfection. The data are shown as mean ± SD of triplicate experiments. A two-way analysis of variance (ANOVA), followed by post hoc Tukey’s HSD test, were used to test statistical differences between the studied groups. A p values less than 0.05 were considered as significant. All bars on the charts, which are marked with the same letter (even if it is only one common letter among many), are not significantly different