A2780, SKOV-3 and OV-90 EOC cell lines were used. The A2780 cells originate from a human patient with primary EOC without chemotherapy treatment, and were obtained from the European Collection of Authenticated Cell Cultures. The SKOV-3 cells are derived from the ascites of a patient with EOC, whereas the OV-90 cells correspond to high-grade human EOC cell lines originating from metastases; these two cell lines were acquired from the American Type Culture Collection.

A2780, SKOV-3 and OV-90 cells were cultured in RPMI 1640 medium (Gibco; Thermo Fischer Scientific, Inc.) supplemented with 10% charcoal/dextran treated fetal bovine serum (FBS; HyClone; Cytiva) at 37°C with 5% CO₂. Once the cells reached 80% confluence, they were plated for transfection.

Cell lines were transfected with miR-145-5p mimics (30 nM; sense, 5′-GUCCAGUUUUCCCAGGAAUCCCU-3′ and antisense, 5′-AGGGAUUCCUGGGAAAACUGGAC-3′), miR-23b-3p mimics (30 nM; sense, 5′-AUCACAUUGCCAGGGAUUACC-3′ and antisense, 5′-GGUAAUCCCUGGCAAUGUGAU-3′) and a mixture of both miRs at 30 nM concentrations (Integrated DNA Technologies, Inc.) using Viafect transfection reagent (cat. no. E498A; Promega Corporation) at a concentration of 2 µl/ml according to the manufacturer's protocol. Transfection was conducted at 37°C for 48 h (or the time indicated for each experiment) in RPMI 1640 medium supplemented with 2% FBS. As a negative control, cells were transfected with a scrambled sequence (Sc; cat. no. SC-37007; Santa Cruz Biotechnology, Inc.) at a final concentration of 10 nM under the aforementioned conditions. For the control condition, cells were maintained at 37°C for 48 h (or the time indicated for each experiment) in RPMI with 2% FBS and Viafect transfection reagent at a concentration of 2 µl/ml. The transfection efficiency was assessed by measuring the expression levels of each miR using reverse transcription-quantitative PCR (RT-qPCR).

A total of 500,000 cells were seeded in 6-well plates and transfected as aforementioned. Extraction of miRs was carried out using chemical and mechanical lysis using QIAzol lysis reagent (Qiagen, Inc.), followed by the miRNeasy kit (Qiagen, Inc.), according to the manufacturer's instructions. The miScript II RT kit (cat. no. 218161; Qiagen, Inc.) was used for the reverse transcription of RNA into cDNA, adhering to the manufacturer's protocol for miRs.

To detect miR-145 and miR-23b levels, the miScript SYBR Green qPCR Kit (cat. no. 218076; Qiagen, Inc.) was used in a StepOne Real-Time PCR thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). Ready-to-use miR-145 (5′-GUCCAGUUUUCCCAGGAAUCCCU-3′) and miR-23b (5′-AUCACAUUGCCAGGGAUUACC-3′) forward primers were selected for qPCR (cat. nos. MS00003528 and MS00031647; Qiagen, Inc.). Small nuclear RNA U6 (forward primer, 5′-CGCTTCGGCAGCACATATACTA-3′; cat. no. MS00033740; Qiagen, Inc.) was used as a miR housekeeping control. The reverse primer is included in the miScript SYBR Green qPCR kit (cat. no. 218076; Qiagen, Inc.) and is designed specifically for this purpose. Nuclease-free water was used as a negative control. The thermocycling conditions were as follows: Initial denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 30 sec and extension at 70°C for 30 sec. mRNA levels were quantified using the 2^−∆∆Cq method (42).

EOC cells were seeded in 24-well plates (50,000 cells/well) containing round coverslips. Cells were transfected with miR-145, miR-23b or the combination as aforementioned. After 48 h, cells were washed with 1X PBS and subsequently fixed with 4% paraformaldehyde in 1X PBS for 15 min at room temperature. Cells were then permeabilized with 0.3% Triton X-100 for 10 min and blocked with PBS containing 2% BSA (MilliporeSigma) for 5 min at room temperature, to prevent non-specific binding of the primary antibody. Samples were incubated overnight at 4°C with anti-Ki-67 mouse monoclonal primary antibody (cat. no. SC-23900; Santa Cruz Biotechnology, Inc.) at a 1:300 dilution in PBS containing 2% BSA. The secondary antibody used was Goat anti-Mouse IgG Alexa Fluor 594 (cat. no. A11005; Molecular Probes; Thermo Fisher Scientific, Inc.), diluted to 1:200 in PBS containing 2% BSA and incubated for 1 h at 37°C. ProLong Gold Antifade with DAPI (cat. no. 8961S; Cell Signaling Technology, Inc.) was used at room temperature for 24 h to mount the coverslips, allowing visualization of cell nuclei. Immunofluorescence staining was detected using a fluorescence microscope (Olympus BX51TF; Olympus Corporation). The stained area from 4–6 fields of view was evaluated per condition using Image Pro-Plus 6.2 software (Media Cybernetics, Inc.).

Cell counting was carried out using the images obtained from the Ki-67 immunofluorescence assays, conducted as aforementioned, using Fiji ImageJ software (version 1,53e; National Institutes of Health). For each condition, 4–6 fields of view were analyzed using fluorescence microscopy.

A total of 800,000 cells were seeded in 6-well plates to evaluate the effect of miR-145 and miR-23b co-transfection on the migration of EOC cells. Cells were transfected as aforementioned for 48 h (A2780 cells) or 4 h (SKOV-3 and OV-90 cells). After the transfection period, migration was evaluated using the BioCoat kit (Corning, Inc.). A total of ~100,000 EOC cells transfected with miRs and previously resuspended in RPMI without FBS, were introduced into the upper chamber of the inserts, which were coated on the bottom with 0.003 mg/ml fibronectin (for A2780 and SKOV-3 cells) or 0.01 mg/ml fibronectin (for OV-90 cells). The inserts were placed in RPMI medium with 10% FBS, serving as a cell attractant. EOC cells were allowed to migrate for a duration that was shorter than their respective cell doubling period to evaluate their migratory potential (43–45). A2780 cells were allowed to migrate at 37°C for 8 h, whereas SKOV-3 and OV-90 cells were allowed to migrate at 37°C for 4 and 24 h, respectively. Cells that migrated were fixed and stained with 0.1% crystal violet in 99.8% methanol for 1 h at room temperature. Cells that crossed the insert and remained attached to the lower membrane surface were observed under a light microscope (Olympus BX51TF; Olympus Corporation) and 6–8 images for each experimental condition were obtained. The cells in each image were counted using the Fiji ImageJ program. The effect of the transfection reagent only (Control) and Sc on the migration of EOC cells was determined to corroborate that no significant differences existed between these conditions. The Control condition was chosen as the control for all of the results.

A total of 800,000 EOC cells were seeded in 6-well plates and transfected as described in the transient transfection and evaluation of cell migration sections. To assess cell invasion, the commercial BioCoat kit (Corning, Inc.) was used, comprising Matrigel-coated Transwell inserts. The Matrigel inserts were hydrated for 2 h at 37°C with RPMI medium without FBS prior to use. Subsequently, the inserts were positioned in a 24-well plate containing RPMI medium with 10% FBS as an attractant. A total of 100,000 previously transfected cells in RPMI medium without FBS were added to the upper chamber of the insert. EOC cells were allowed to invade for a shorter time period than their respective cell doubling time to evaluate invasion (41–43). The invasion assay was carried out for 16 h (SKOV-3 cells) or 24 h (A2780 and OV-90 cells) at 37°C. Invading cells were fixed in methanol at −20°C for 2 min and then stained with 1% toluidine blue overnight at room temperature. The inserts were observed under a light microscope (Olympus BX51TF; Olympus Corporation) to obtain images (6–8 images for each experimental condition) of cells attached to the lower surface of the membrane (cells that crossed the insert). Images were analyzed using the Fiji ImageJ program. The effect of the Control and Sc on the invasion of EOC cells was determined to corroborate that there were no significant differences between these conditions.

To obtain proteins, 500,000 EOC cells were seeded and transfected for 48 h with miR-145 and miR23-b, as described in the transfection section. Protein extraction was conducted using RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1% Triton X-100; pH 8.0) and a 1X protease and phosphatase inhibitor cocktail (cat. no. 1861284; Thermo Fisher Scientific, Inc.). The cells were scraped on ice for 5 min and the lysate was sonicated (2 times for 5 sec each) on ice to complete lysis. The BCA Protein Assay kit (cat. no. 23227; Thermo Fisher Scientific, Inc.) was used for protein quantification.

Western blotting was performed as previously described (14). The protocol involved electrophoresis of proteins (c-MYC, 50 µg of protein for OV-90 and A2780 cells, and 80 µg of protein for SKOV-3 cells was loaded; ZEB1, 50 µg of protein for SKOV-3 and A2780 cells, and 80 µg of protein was loaded for OV-90 cells; and ABCB1, a total of 80 µg of protein was loaded), followed by wet transfer to a 0.45-µm nitrocellulose membrane (cat. no. NBA0858001EA; PerkinElmer, Inc.,). The membranes were blocked with 5% non-fat dry milk in Tris-Buffered Saline containing 0.1% Tween 20 at room temperature for 1 h, and then incubated with c-MYC (1:350; cat. no. D84C12; Cell Signaling Technology, Inc.), ZEB1 (1:350; cat. no. E2G6Y; Cell Signaling Technology, Inc.), ABCB1 (1:250; cat. no. D3H1Q; Cell Signaling Technology, Inc.) and β-actin (1:10,000; Merck KGaA; cat. no. A5441) primary antibodies at 4°C overnight with constant shaking. The membranes were then incubated with secondary antibodies coupled to HRP while shaking at room temperature for 1 h. Anti-rabbit (1:5,000; cat. no. 111-035-003; Jackson ImmunoResearch Laboratories, Inc.) or anti-mouse (1:500; cat. no. 115-035-003; Jackson ImmunoResearch Laboratories, Inc.) antibodies were used. Protein bands were developed using Westar Sun ECL detection reagent (cat. no. XLS063.02500; Cyanagen Srl). Chemiluminescent signals on the membrane were detected with the Image Quant LAS 500 equipment (GE Healthcare Life Sciences). The Fiji ImageJ program (National Institutes of Health) was used to semi-quantify the bands and conduct a densitometric analysis of band intensity. The results were expressed in arbitrary units. The results from each sample were normalized to the intensity of the corresponding β-actin housekeeping protein band. A molecular weight marker (cat. no. 02102-250; MaestroGen, Inc.) was used as a reference. No significant differences in protein levels were detected between the Control and Sc conditions. Therefore, the semi-quantification results of western blotting were analyzed in relation to the Control.

GraphPad Prism 8.0.2 for Windows (Dotmatics) was used for the statistical analysis. The Kruskal-Wallis test and Dunn's post hoc test were used to analyze the differences among groups. Data are presented as the mean ± SEM. P<0.05 was considered to indicate a statistically significant difference. For all the experiments, n=3 was the minimum number of repeats for statistical analysis, and n=4 was performed where specified in figures.

Transient transfection was used to assess the incorporation of the miRs into EOC cells. The transfection efficiency was evaluated using RT-qPCR. As no significant differences were observed between the Sc and Control groups, all results were compared with the Control group.

A significant increase in miR-145 and miR-23b expression was observed in all ovarian cell lines after transfection with the miR itself or the combination of miRs (Mix) (Fig. 1). The transfection of A2780 cells with miR-145 or miR-23b alone, as well as the co-transfection of both miRs, significantly decreased the levels of Ki-67 (Fig. 2A and B). Conversely, Ki-67 levels were significantly decreased only when overexpressing miR-23b and the miR mix in SKOV-3 (Fig. 2C and D) and OV-90 (Fig. 2E and F) cells; thus, the combination of both miRs decreased Ki-67 marker levels across all three cell lines, resulting in reductions of 49, 37 and 33% in A2780, SKOV-3 and OV-90 cells, respectively (Fig. 2).

Consistent with aforementioned results, Ki-67-positive A2780 cells (marked in red in Fig. 2A) were reduced by 43, 53 and 57% after the transfection of miR-145, miR-23b and the miR mix, respectively (Fig. 2G). In SKOV-3 cells, Ki-67-positive cells were decreased only following miR-23b (32%) and miR Mix (29%) transfection compared with the control group, while Ki-67-positive OV-90 cells were decreased by 34 and 42% following transfection with miR-23b and Mix (Fig. 2H and I). The findings from Ki-67 immunofluorescence staining and Ki-67-positive cell counting indicated that co-transfection of miR-145 and miR-23b decreased the proliferation of EOC cells.

Cell migration was reduced by 29, 23 and 44% in A2780 cells after the transient overexpression of miR-145, miR-23b and the miR Mix, respectively, compared with the Control condition. In addition, the miR Mix significantly decreased cell migration compared with the overexpression of miR-23b only (Fig. 3A and D). Similarly, the overexpression of miR-145, miR-23b and miR Mix reduced the migration of SKOV-3 cells. Furthermore, the miR Mix significantly decreased cell migration compared with that of cells transfected with miR-145 alone (Fig. 3B and E). OV-90 cells exhibited a significant decrease in cell migration after transfection with miR-145, miR-23b and the miR Mix. Notably, OV-90 cells transfected with the miR Mix also exhibited a significant decrease in cell migration compared with those that were transfected with miR-145 or miR-23b alone (Fig. 3C and F).

Transfection of cells with the miR Mix decreased the invasion of A2780 cells by >80% (Fig. 4A and B). Furthermore, invasion decreased by ~55 and 44% in SKOV-3 and OV-90 cells, respectively, relative to the control (Fig. 4C-F). Thus, these results indicated that the combination of miR-145 and miR-23b decreased the migration and invasion of EOC cells.

A2780 cells exhibited decreased c-MYC levels after transfection with miR-145, miR-23b or the miR Mix (Fig. 5A and B). In particular, Mix reduced c-MYC levels by 42% compared with the control. c-MYC levels were significantly decreased in SKOV-3 cells (Fig. 5C and D) and OV-90 cells (Fig. 5E and F) after transfection with miR-23b or the miR Mix. These results indicated that miR-145 and miR-23b co-transfection decreased c-MYC levels in all three EOC cell lines.

ZEB1 is a transcription factor with a predicted molecular weight of 125 kDa; however, several studies have reported that the actual molecular weight of this protein ranges between 125 and 200 kDa (46–48). Similarly, in the present study, bands between 125 and 200 kDa were observed in EOC cells (Fig. 6). The results in A2780 cells revealed that ZEB1 expression was decreased by 29% when overexpressing miR-145, by 39% when overexpressing miR-23b and by 41% when overexpressing both miRs (Fig. 6A and B). In SKOV-3 cells, ZEB1 expression decreased by 35 and 41% following transfection with miR-23b and the Mix, respectively (Fig. 6C and D). In OV-90 cells, ZEB1 expression decreased by 46, 29 and 60% following transfection with miR-145, miR-23b and the Mix, respectively (Fig. 6E and F).

ABCB1 expression was reduced by 34% in A2780 cells when overexpressing miR-145 and miR-23b separately; however, co-transfection with both miRs resulted in a 56% reduction in ABCB1 expression (Fig. 7A and B). In SKOV-3 cells, ABCB1 expression was decreased by 40, 63 and 78% following transfection with miR-145, miR-23b and the Mix, respectively (Fig. 7C and D). These results indicated that the co-transfection of miR-23b and miR-145 decreased the levels of the drug efflux transporter ABCB1 in A2780 and SKOV-3 cell lines. Since OV-90 cells have low levels of ABCB1 at baseline, ABCB1 levels were assessed only in A2780 and SKOV-3 cells.