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miR‑145 and miR‑23b co‑transfection decreases proliferation, migration, invasion and protein levels of c‑MYC, ZEB1 and ABCB1 in epithelial ovarian cancer cell lines

  • Authors:
    • Allison Fredes‑Garrido
    • Álvaro Armijo Cruz
    • Gloria M. Calaf
    • Maritza P. Garrido
    • Carmen Romero
  • View Affiliations / Copyright

    Affiliations: Laboratory of Endocrinology and Reproductive Biology, Clinical Hospital University of Chile, Santiago 8380456, Chile, Institute of Advanced Research, University of Tarapacá, Arica 1000000, Chile
    Copyright: © Fredes‑Garrido et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 246
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    Published online on: July 8, 2025
       https://doi.org/10.3892/mmr.2025.13611
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Abstract

MicroRNAs (miRs) are non‑coding RNAs that prevent the translation of mRNAs. miRs participate in cellular processes such as cell proliferation, migration and invasion, acting as oncogenes or tumor suppressors. In epithelial ovarian cancer (EOC), a decrease in tumor suppressor miRs, such as miR‑145 and miR‑23b, regulates the mRNAs of oncogenic proteins. The present study aimed to determine whether the co‑transfection of two oncosuppressor miRs (miR‑145‑5p and miR‑23b‑3p) decreased the proliferation, migration, invasion, and protein levels of c‑MYC, zinc finger E‑box binding homeobox 1 (ZEB1) and ATP binding cassette subfamily B1 (ABCB1) in EOC cell lines (A2780, SKOV‑3 and OV‑90). Reverse transcription‑quantitative PCR was employed to determine miR expression after co‑transfection. Cell proliferation was evaluated by Ki‑67 immunofluorescence staining and Ki‑67 positive cell counting. Transwell inserts, both with and without Matrigel, were used to assess invasion and migration, respectively. c‑MYC, ZEB1 and ABCB1 protein expression was determined by western blot analysis. The co‑transfection of miR‑145 and miR‑23b resulted in decreased proliferation, migration and invasion, along with reduced protein expression levels of c‑MYC, ZEB1 and ABCB1 in EOC cells. The combination of miR‑23b and miR‑145 transfection in EOC cells exhibited good antitumor effects, thus supporting the design of future complementary therapies for EOC.
View Figures

Figure 1

Expression levels of miR-145 and
miR-23b after transfection of ovarian cancer cells. A2780, SKOV-3
and OV-90 cells were transfected with miR-145, miR-23b, Mix, Sc or
Control using Viafect reagent for 48 h at 37°C. RNU6 was used as
the control to quantify mRNA levels. n=3 for A2780, SKOV and OV-90
cells. *P<0.05 and **P<0.01 vs. Control (Kruskal-Wallis test
with Dunn's post hoc test). Data are presented as the mean ± SEM.
miR, microRNA; Mix, combination of miR-145 and miR-23b; Sc,
scrambled sequence; RNU6, small nuclear RNA U6.

Figure 2

Effect of miR-145 and miR-23b
co-transfection on the proliferation of EOC cells. Cell
proliferation was assessed using Ki-67 immunofluorescence staining
and EOC cell quantification was conducted using Ki-67
immunofluorescence images (red cells). Representative
immunofluorescence images of Ki-67 (red) in (A) A2780, (C) SKOV-3
and (E) OV-90 cells. Magnification, ×400; scale bar, 50 µm.
Semi-quantitative analysis of Ki-67 immunofluorescence in (B)
A2780, (D) SKOV-3 and (F) OV-90 cells was conducted using Image
Pro-Plus 6.2 computer software. Data for semi-quantification of
fluorescence were normalized to the Control condition. n=4 (4–6
images per condition). Semi-quantitative analysis of Ki-67-positive
EOC cells (red) in (G) A2780, (H) SKOV-3 and (I) OV-90 cells was
carried out using the Fiji ImageJ program. n=4 (3–4 images per
condition were analyzed; magnification, ×400). Conditions were
normalized to the Control. *P<0.05, **P<0.01 and
***P<0.001 compared vs. Control (Kruskal-Wallis test with Dunn's
post hoc test). Data are presented as the mean ± SEM. miR,
microRNA; EOC, epithelial ovarian cancer; Mix, combination of
miR-145 and miR-23b; Sc, scrambled sequence.

Figure 3

Effect of miR-145 and miR-23b
co-transfection on epithelial ovarian cancer cell migration.
Migration was evaluated using Transwell inserts. Representative
images of post-treatment migration assays in (A) A2780, (B) SKOV-3
and (C) OV-90 cells. Scale bar, 50 µm. Semi-quantitative analysis
of migration data in (D) A2780, (E) SKOV-3 and (F) OV-90 cells.
Changes are presented relative to the Control. n=3 (6–8 images per
condition). *P<0.05, **P<0.01 and ***P<0.001 vs. Control
(Kruskal-Wallis test followed by Dunn's post hoc test).
aP<0.05 and aaP<0.01 vs. Mix
(Kruskal-Wallis test followed by Dunn's post hoc test). Data are
presented as the mean ± SEM. miR, microRNA; Mix, combination of
miR-145 and miR-23b; Sc, scrambled sequence.

Figure 4

Effect of miR-145 and miR-23b
co-transfection on epithelial ovarian cancer cell invasion. The
invasion assay was carried out using Matrigel-coated Transwell
inserts. Representative images of invasion assays following
treatment with miRs at 37°C for 24 h in (A) A2780 and (E) OV-90
cells, and (C) 16 h in SKOV-3 cells. Scale bar, 50 µm.
Semi-quantitative analysis of images of invasion data in (B) A2780,
(D) SKOV-3 and (F) OV-90 cells. Data are presented as multiples of
change relative to the Control. n=3 (10 images per condition).
***P<0.001 vs. Control condition (Kruskal-Wallis test followed
by Dunn's post hoc test). Data are presented as the mean ± SEM.
miR, microRNA; Mix, combination of miR-145 and miR-23b; Sc,
scrambled sequence.

Figure 5

Western blot analysis of c-MYC in EOC
cells. Representative images c-MYC protein expression (57–65 kDa)
in (A) A2780, (C) SKOV-3 and (E) OV-90 cells. A total of 50 µg of
protein (A2780 and OV-90 cells) and 80 µg protein (SKOV-3 cells)
were loaded, and β-actin (42 kDa) was used as a loading control.
Semi-quantitative analysis of the western blotting bands in EOC
cells after transfection under different conditions in (B) A2780,
(D) SKOV-3 and (F) OV-90 cells. Data were normalized to β-actin and
are presented as the fold change compared with the Control. n=4.
*P<0.05, **P<0.01 and ***P<0.001 vs. Control
(Kruskal-Wallis test followed by Dunn's post hoc test). Data are
presented as the mean ± SEM. miR, microRNA; EOC, epithelial ovarian
cancer; Mix, combination of miR-145 and miR-23b; Sc, scrambled
sequence; AU, arbitrary units.

Figure 6

Western blot analysis of ZEB1
expression in EOC cells. Representative images of ZEB1 protein
expression (125–200 kDa) in (A) A2780, (C) SKOV-3 and (E) OV-90 EOC
cells. A total of 50 µg (A2780 and SKOV-3 cells) or 80 µg (OV-90
cells) of protein were loaded. β-actin (42 kDa) was used as a
loading control. Semi-quantitative analysis of the western blotting
bands in (B) A2780, (D) SKOV-3 and (F) OV-90 cells after
transfection under different conditions. Values were normalized to
β-actin and expressed as the fold change compared with the Control.
n=4. *P<0.05, **P<0.01 and ***P<0.001 vs. Control
(Kruskal-Wallis test followed by Dunn's post hoc test). Data are
presented as the mean ± SEM. miR, microRNA; EOC, epithelial ovarian
cancer; Mix, combination of miR-145 and miR-23b; Sc, scrambled
sequence; AU, arbitrary units; ZEB1, zinc finger E-box binding
homeobox 1.

Figure 7

Western blot analysis of ABCB1 in
A2780 and SKOV-3 cells. Representative images of ABCB1 (130–180
kDa) protein expression in (A) A2780 and (C) SKOV-3 cells. A total
of 80 µg protein was loaded. β-actin (42 kDa) was used as the
loading control. Semi-quantitative analysis of western blot bands
in (B) A2780 and (D) SKOV-3 cells post-transfection under the
different conditions. Values were normalized to β-actin and the
Control. n=4. *P<0.05, **P<0.01 and ***P<0.001 vs. Control
(Kruskal-Wallis test followed by Dunn's post hoc test). Data are
presented as the mean ± SEM. miR, microRNA; Mix, combination of
miR-145 and miR-23b; Sc, scrambled sequence; AU, arbitrary units;
ABCB1, ATP binding cassette subfamily B1.

Figure 8

Effect of miR-145 and miR-23b
co-transfection in ovarian cancer cells. NGF binds to its
high-affinity receptor TRKA, thereby activating MAPK and PI3K/AKT
signaling pathways, which decreases the transcription of miR-145
and miR-23b, and increases oncogenic proteins involved in the
progression of ovarian cancer. Restoring miR-145 and miR-23b levels
in epithelial ovarian cancer cells decreases the levels of the
oncogenic proteins c-MYC, ZEB1 and ABCB1, which are involved in
proliferation, invasion, migration and chemoresistance. miR,
microRNA; NGF, nerve growth factor; TRKA, tropomyosin kinase A
receptor; ZEB1, zinc finger E-box binding homeobox 1; ABCB1, ATP
binding cassette subfamily B1.
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Copy and paste a formatted citation
Spandidos Publications style
Fredes‑Garrido A, Cruz Á, Calaf GM, Garrido MP and Romero C: miR‑145 and miR‑23b co‑transfection decreases proliferation, migration, invasion and protein levels of c‑MYC, ZEB1 and ABCB1 in epithelial ovarian cancer cell lines. Mol Med Rep 32: 246, 2025.
APA
Fredes‑Garrido, A., Cruz, Á., Calaf, G.M., Garrido, M.P., & Romero, C. (2025). miR‑145 and miR‑23b co‑transfection decreases proliferation, migration, invasion and protein levels of c‑MYC, ZEB1 and ABCB1 in epithelial ovarian cancer cell lines. Molecular Medicine Reports, 32, 246. https://doi.org/10.3892/mmr.2025.13611
MLA
Fredes‑Garrido, A., Cruz, Á., Calaf, G. M., Garrido, M. P., Romero, C."miR‑145 and miR‑23b co‑transfection decreases proliferation, migration, invasion and protein levels of c‑MYC, ZEB1 and ABCB1 in epithelial ovarian cancer cell lines". Molecular Medicine Reports 32.3 (2025): 246.
Chicago
Fredes‑Garrido, A., Cruz, Á., Calaf, G. M., Garrido, M. P., Romero, C."miR‑145 and miR‑23b co‑transfection decreases proliferation, migration, invasion and protein levels of c‑MYC, ZEB1 and ABCB1 in epithelial ovarian cancer cell lines". Molecular Medicine Reports 32, no. 3 (2025): 246. https://doi.org/10.3892/mmr.2025.13611
Copy and paste a formatted citation
x
Spandidos Publications style
Fredes‑Garrido A, Cruz Á, Calaf GM, Garrido MP and Romero C: miR‑145 and miR‑23b co‑transfection decreases proliferation, migration, invasion and protein levels of c‑MYC, ZEB1 and ABCB1 in epithelial ovarian cancer cell lines. Mol Med Rep 32: 246, 2025.
APA
Fredes‑Garrido, A., Cruz, Á., Calaf, G.M., Garrido, M.P., & Romero, C. (2025). miR‑145 and miR‑23b co‑transfection decreases proliferation, migration, invasion and protein levels of c‑MYC, ZEB1 and ABCB1 in epithelial ovarian cancer cell lines. Molecular Medicine Reports, 32, 246. https://doi.org/10.3892/mmr.2025.13611
MLA
Fredes‑Garrido, A., Cruz, Á., Calaf, G. M., Garrido, M. P., Romero, C."miR‑145 and miR‑23b co‑transfection decreases proliferation, migration, invasion and protein levels of c‑MYC, ZEB1 and ABCB1 in epithelial ovarian cancer cell lines". Molecular Medicine Reports 32.3 (2025): 246.
Chicago
Fredes‑Garrido, A., Cruz, Á., Calaf, G. M., Garrido, M. P., Romero, C."miR‑145 and miR‑23b co‑transfection decreases proliferation, migration, invasion and protein levels of c‑MYC, ZEB1 and ABCB1 in epithelial ovarian cancer cell lines". Molecular Medicine Reports 32, no. 3 (2025): 246. https://doi.org/10.3892/mmr.2025.13611
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