MicroRNA‑195 suppresses cell proliferation, migration and invasion in epithelial ovarian carcinoma via inhibition of the CDC42/CCND1 pathway

Abstract

Epithelial ovarian carcinoma (EOC) is the most common cause of gynecological cancer mortality, and poses a threat to women. MicroRNA‑195 (miR‑195) has been reported to induce apoptosis of human OVCAR‑3 cells by inhibiting the VEGFR2/AKT pathway. However, the role of miR‑195 in EOC remains unknown. A previous study reported that cell division cycle 42 (CDC42) can serve as a target gene of miR‑195 and mediate malignant progression of esophageal squamous cell carcinoma (ESCC). The aim of the present study was to investigate the role of miR‑195 in EOC and the regulation in CDC42/CCND1 pathway. Tissues samples and clinical materials were collected from 78 enrolled patients with EOC to analyze the expression and clinical significance of miR‑195, CDC42 and cyclin D1 (CCND1). Human EOC cell lines OVCA420, OVCAR‑3, A2780 and SKOV3 cell lines were used to assess the expression and function of miR‑195, CDC42 and CCND1 in vitro. Cell proliferation, the cell cycle and apoptosis, as well as the cell migratory and invasive abilities were detected in vitro using BrdU incorporation, colony formation, wound healing and Transwell invasion assays, along with flow cytometry. miR‑195 was downregulated, while CDC42 and CCND1 were upregulated in human EOC tissues and cells, and the aberrant expression of both was associated with increased EOC malignancy. Moreover, miR‑195 expression was negatively correlated with CDC42 and CCND1 expression levels, and negatively regulated these expression levels. Thus, it was suggested that miR‑195 functions as a tumor suppressor, but CDC42 and CCND1 act as tumor promoters based their abilities to enhance cell proliferation, cell cycle entry, migration and invasion, as well as decrease apoptosis in OVCAR‑3 cells. the present results demonstrated that miR‑195 inhibited human EOC progression by downregulating CDC42 and CCND1 expression. Furthermore, it was identified that miR‑195, CDC42 and CCND1 may be effective biomarkers for EOC diagnosis and treatment.

Figure 1

miR-195 is downregulated, while CDC42 and CCND1 are upregulated in human EOC tissues and cells. (A) mRNA expression of miR-195 in human EOC tissues. (B) Protein expression levels of CDC42 and CCND1 in human EOC tissues. (C) Immunohistochemical staining of CDC42 and CCND1 in human EOC tissues. Scale bar, 100 µm. (D) Protein expression levels of CDC42 and CCND1 in human EOC cells. (E) mRNA expression of miR-195 in diverse human EOC cells. Data in Fig. 1A-C were analyzed using a paired t-test. ^*P<0.05, ^**P<0.01 vs. healthy tissues. Data in the Fig. 1D were analyzed using a one-way ANOVA. ^*P<0.05 vs. HS832-Tc cells; ^#P<0.05 vs. SKOV3 cells; ^$P<0.05 vs. OVCA420 cells; ^@P<0.05 vs. A2780 cells. EOC, epithelial ovarian carcinoma; miR, microRNA; CDC42, Cell division cycle 42; CCND1, cyclin D1.

Figure 2

miR-195 can regulate CDC42 and CCND1 expression levels. Correlation between miR-195 and (A) CDC42 and (B) CCND1 expression levels in human epithelial ovarian carcinoma tissues were analyzed using Pearson's correlation coefficient. (C) mRNA expression of miR-195 in OVCAR-3 cells transfected with miR-195 mimic, mimic-NC, miR-195 inhibitor and inhibitor-NC. mRNA expression levels of (D) CDC42 and (E) CCND1 with overexpression or knockdown of miR-195 in OVCAR-3 cells. Data were analyzed using one-way ANOVA. ^*P<0.05. miR, miR-195; Mim, mimic; NC, negative control; Inh, inhibitor; CDC42, Cell division cycle 42; CCND1, cyclin D1.

Figure 3

miR-195 inhibits cell proliferation by reducing CDC42 and CCND1 expression levels in OVCAR-3 cells in vitro. mRNA expression levels of (A) CDC42 and (B) CCND1 in OVCAR-3 cells transfected with miR-195 inhibitor, inhibitor-NC, miR-195 inhibitor + NC and miR-195 inhibitor + CDC42 or CCND1 siRNAs. mRNA expression levels of (C) CDC42 and (D) CCND1 in OVCAR-3 cells transfected with miR-195 mimic, mimic-NC, miR-195 mimic + NC-vector and miR-195 mimic + CDC42 or CCND1 pcDNA. (E) Cell proliferation was assessed using BrdU immunostaining at 24 h after transfection. Red, OVCAR-3 cells labeled with BrdU; blue, nuclei counterstained by BrdU. Scale bar, 50 µm. (F) Evaluation of cell proliferation via colony formation assays at 48 h after transfection. Data were analyzed using one-way ANOVA. ^*P<0.05 vs. Inh-NC or Mim-NC groups; ^#P<0.05 vs. miR Inh-siNC or miR Mim + NC-Vector groups. BrdU, 5-bromo-2-deoxyuridine; miR, miR-195; Inh, inhibitor; NC, negative control, siRNA, short interfering RNA; Mim, mimic; CDC42, Cell division cycle 42; CCND1, cyclin D1.

Figure 4

miR-195 inhibits cell cycle entry in OVCAR-3 cells by downregulating CDC42 and CCND1 expression levels in vitro. (A) Effects of miR-195, CDC42 and CCND1 on the percentage of PI-stained OVCAR-3 cells at G₁, S and G₂ phases. (B) Cell cycle of each group. Data were analyzed via one-way ANOVA. ^*P<0.05 vs. Inh-NC or Mim-NC groups; ^#P<0.05 vs. miR Inh-siNC or miR Mim + NC-Vector groups. miR, miR-195; Inh, inhibitor; NC, negative control, siRNA, short interfering RNA; Mim, mimic; CDC42, Cell division cycle 42; CCND1, cyclin D1.

Figure 5

miR-195 promotes apoptosis in OVCAR-3 cells by downregulating CDC42 and CCND1 expression levels in vitro. (A) Effects of miR-195, CDC42 and CCND1 on cell apoptosis. (B) Apoptotic cells in each group. Data were analyzed using one-way ANOVA. ^*P<0.05 vs. Inh-NC or Mim-NC groups; ^#P<0.05 vs. miR Inh-siNC or miR Mim + NC-Vector groups. miR, miR-195; Inh, inhibitor; NC, negative control, siRNA, short interfering RNA; Mim, mimic; CDC42, Cell division cycle 42; CCND1, cyclin D1.

Figure 6

miR-195 suppresses cell migration and invasion in OVCAR-3 cells by inhibiting CDC42 and CCND1 expression levels in vitro. Effects of miR-195 (A) inhibitor or (B) mimic, CDC42 and CCND1 on OVCAR-3 cell migration using wound healing assay at 0 and 24 h after transfection. Scale bar, 200 µm. (C) Effects of miR-195, CDC42 and CCND1 on OVCAR-3 cell invasion via Transwell invasion assay at 48 h after transfection. Scale bar, 50 µm. Data were analyzed using one-way ANOVA. ^*P<0.05 vs. Inh-NC or Mim-NC groups; ^#P<0.05 vs. miR Inh-siNC or miR Mim + NC-Vector groups. miR, miR-195; Inh, inhibitor; NC, negative control, siRNA, short interfering RNA; Mim, mimic; CDC42, Cell division cycle 42; CCND1, cyclin D1.