MicroRNA-936 targets FGF2 to inhibit epithelial ovarian cancer aggressiveness by deactivating the PI3K/Akt pathway.

PURPOSE: MicroRNA-936 (miR-936) was previously reported to be dysregulated and involved in the development of non-small cell lung cancer and glioma. However, the functional roles of miR-936 in epithelial ovarian cancer (EOC) remain unclear. In this study, we aimed to evaluate miR-936 expression in EOC and investigate its regulatory role in EOC cell behavior.
METHODS: The expression of miR-936 in EOC was measured by RT-qPCR. Cell proliferation, apoptosis, migration, and invasion in vitro, as well as tumor growth in vivo, were determined by CCK-8, flow cytometry, migration and invasion assays, and xenograft models in nude mice, respectively. Bioinformatics analysis, luciferase reporter assays, RT-qPCR, and Western blot analysis were performed to investigate the relationship between miR-936 and fibroblast growth factor 2 (FGF2).
RESULTS: miR-936 expression was significantly downregulated in EOC tissues and cell lines. Low miR-936 expression was found to be correlated with the tumor size, FIGO stage, and lymphatic metastasis in EOC patients. Functional experiments indicated that ectopic miR-936 expression suppressed EOC cell proliferation, migration, and invasion; promoted cell apoptosis; and decreased tumor growth in vivo. In addition, the FGF2 gene was verified to be a direct target of miR-936 in EOC cells. FGF2 expression levels were upregulated in EOC tissues and were inversely correlated with miR-936 expression. Furthermore, effects of FGF2 silencing were similar to those of miR-936 overexpression in EOC cells. Recovered FGF2 expression rescued the miR-936-induced inhibitory effects in EOC cells. Notably, miR-936 was able to deactivate the PI3K/Akt signaling pathway in EOC cells by regulating FGF2 both in vitro and in vivo.
CONCLUSION: Altogether, our findings provided initial evidence that miR-936 inhibits the aggressiveness of EOC cells in vitro and in vivo, at least partially, by targeting FGF2-mediated suppression of the PI3K/Akt pathway. Therefore, the miR-936/FGF2/PI3K/Akt pathway is a promising therapeutic target for the treatment of EOC patients.

Introduction

Ovarian cancer is the second most prevalent type of gynecological malignant tumor and the fifth most common cause of cancer-related deaths in women worldwide.1,2 Approximately 2,04,000 novel cases and 1,25,000 deaths caused by ovarian cancer occur each year globally.3 Epithelial ovarian cancer (EOC), the most common type of ovarian cancer, accounts for about 90% of ovarian cancer cases and 4.2% of all cancer-associated mortalities among women worldwide.4 Currently, surgery combined with chemotherapy is considered the standard treatment regimen for EOC patients.5 Extensive efforts have been directed towards improving diagnosis and treatment for EOC. However, advanced-stage EOC patients have poor prognosis, with a five-year survival rate of less than 25%.6 The poor prognosis of this disease is primarily attributed to aggressive metastasis, recurrence, and chemotherapy resistance.7 Therefore, an in-depth understanding of the molecular mechanisms underlying EOC occurrence and development is crucial for the development of therapeutic approaches to improve patient outcomes.

MicroRNAs (miRNAs) are a class of highly conserved noncoding RNAs that are about 21–25 nucleotides long.8 MiRNAs have been recognized as important regulators of gene expression and act by directly binding to the 3ʹ-untranslated regions (3ʹ-UTRs) of their target genes, leading to messenger RNA (mRNA) degradation or translational suppression.9 Aberrant miRNA expression has been widely described in almost all human cancer types, including EOC.10–12 Accumulating evidence has shown that numerous miRNAs are dysregulated in EOC. These dysregulated miRNAs are closely related to the initiation and progression of EOC by regulating various important physiological processes, such as the cell cycle, cell proliferation, apoptosis, death, and metastasis.13–15 The aberrantly expressed miRNAs are believed to act as tumor suppressors or by exerting oncogenic activity to regulate EOC pathogenesis, and their functions are strongly dependent on their target genes.16 Hence, the identification of miRNAs that can be used as effective therapeutic targets shows potential value for the treatment of EOC patients.

MiR-936 levels have been previously reported to be dysregulated and involved in the development of non-small cell lung cancer17 and glioma.18 However, the functional roles of miR-936 are rarely reported in EOC. In the present study, we hypothesized that miR-936 may dysregulated in EOC, and its dysregulation may be involced in the aggressiveness of EOC. To this end, we aimed to analyze miR-936 expression and determine its clinical significance in EOC. In addition, we explored the functional roles of miR-936 and its underlying mechanism of action in EOC. Our study identified a novel pathway, miR-936/FGF2/PI3K/Akt pathway, involved in EOC progression.

Materials and methods

Clinical samples

A total of 51 pairs of EOC tissues and adjacent normal tissues were collected from patients who underwent surgery between June 2016 and September 2017 at the Yidu Central Hospital of Weifang. None of the patients received chemotherapy, radiotherapy, or any other anti-tumor therapies prior to surgical resection. All tissues were immediately frozen in liquid nitrogen and stored at –80 °C for subsequent analyses. The present study was approved by the Research Ethics Committee of Yidu Central Hospital of Weifang and conducted following the ethical standards of the Declaration of Helsinki. In addition, written informed consent was provided by all enrolled patients or their families.

Cell lines

A total of four human EOC cell lines (OVCAR3, SKOV3, CAOV-3, and ES-2) and a human normal ovarian epithelial cell line (NOEC) were purchased from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin mixture (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.

Transfection assay

MiR-936 mimics, corresponding scrambled control (miR-control), small interfering RNA (siRNA) targeting the FGF2 gene (si-FGF2), and negative control siRNA (si-NC) were obtained from GenePharma Co. Ltd. (Shanghai, China). FGF2-overexpression plasmid pcDNA3.1-FGF2 and pcDNA3.1 empty plasmid were chemically synthesized by GeneCopoeia Co. Ltd. (Guangzhou, China). Cells were plated into 6-well plates at a density of 5×105 cells/well. Cells were incubated overnight and then transfected with the aforementioned molecular products using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from tissues or cells using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. Total RNA was reverse-transcribed into complementary DNA (cDNA) for miR-936 and FGF2 mRNA analysis using a miScript Reverse Transcription kit (Qiagen GmbH, Hilden, Germany) and a PrimeScript RT Reagent Kit (TaKaRa, Tokyo, Japan), respectively. Then, mRNA levels of miR-936 and FGF2 were quantified using a miScript SYBR Green PCR kit (Qiagen GmbH, Hilden, Germany) and a SYBR Premix Ex Taq (TaKaRa, Tokyo, Japan), respectively. Small nuclear U6 RNA was used as the internal reference for miR-936, while glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the internal control for FGF2. Relative gene expression was calculated using the 2−∆∆Cq method.19

Cell counting kit 8 (CCK-8) assay

At 48 h after transfection, cells were collected and plated at 3×103 cells/well in 96-well plates. Cells were then incubated at 37 °C in a humidified atmosphere with 5% CO2 for different time periods (0, 24, 48, and 72 h). CCK-8 assay was performed at the indicated time points to determine cellular proliferative ability. In detail, each well was added with 10 µL of CCK-8 reaction reagent (Beyotime Institute of Biotechnology, Shanghai, China) and further incubated at 37 °C with 5% CO2 for 2 h. Finally, an enzyme-linked immunosorbent assay reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to measure the absorbance of each well at 450 nm. Absorbance value is proportional to the proliferative ability of cells. Each assay was performed in triplicate and repeated thrice.

Flow cytometry assay

Cell apoptosis was evaluated using an Annexin V fluorescein isothiocyanate (FITC) apoptosis detection kit (Biolegend, San Diego, CA, USA). Transfected cells were detached using 0.25% trypsin without ethylenediaminetetraacetic acid (EDTA), washed thrice with ice-cold PBS, and then resuspended in 100 µL of 1× binding buffer. Afterwards, cells were stained with 5 µL of Annexin V and 5 µL of PI at room temperature under dark conditions for 30 min. A flow cytometer (FACScan; BD Biosciences, Bedford, MA, USA) was used to examine and measure the number of stained cells.

Migration and invasion assays

A 24-well transwell chamber system (BD Biosciences, Franklin Lakes, NJ, USA) with a Matrigel-coated polycarbonic membrane was used to evaluate cell invasiveness. Non-Matrigel coated chambers were used for the migration assay. For both assays, 5×104 transfected cells suspended in 200 µL of FBS-free medium were plated onto the upper chambers, whereas 500 µL of DMEM containing 20% FBS was added to the lower chambers. After 24 h of incubation at 37 °C with 5% CO2, the migrated and invaded cells were fixed with 4% polyoxymethylene, stained with 0.1% crystal violet, and imaged under an inverted light microscope (CKX41; Olympus Corporation, Tokyo, Japan). The migratory and invasive activities were assessed by counting the number of migrated and invaded cells in five randomly selected fields.

Xenograft tumors in nude mice

All animal experiments were approved by the Institutional Animal Care and Use Committee of Yidu Central Hospital of Weifang and carried out in accordance with the guidance of the Animal Protection Law of the People’s Republic of China-2009 for experimental animals. BALB/c nude mice (six weeks old) were purchased from Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China) and maintained under the following specific pathogen-free conditions: 20–26 °C, 12 h light-dark cycle, and aseptic food and water. All nude mice were subcutaneously injected with EOC cells overexpressing miR-936 or cells in the miR-control group. The width and length of tumors that formed in the nude mice were measured once every four days using Vernier calipers. The tumor volume was calculated using the following formula: volume = (length × width2)/2. All nude mice were sacrificed by cervical dislocation at four weeks after inoculation, and tumor xenografts were removed and weighed.

Bioinformatics analysis

Three online miRNA target prediction software, namely, TargetScan 7.1 (http://www.targetscan.org/), miRanda (http://www.microrna.org), and miRDB (http://www.mirdb.org/), were used to search for the putative targets of miR-936.

Luciferase reporter assay

The fragments containing putative wild-type (wt) or mutant (mut) binding sites for miR-936 in the 3ʹ-UTR of FGF2 were amplified by GenePharma Co. Ltd. and cloned into the psiCHECK™2 vector luciferase reporter vector (Promega Corporation, Madison, WI, USA). The generated luciferase reporter plasmids were defined as psiCHECK-wt-FGF2-3ʹ-UTR and psiCHECK-mut-FGF2-3ʹ-UTR, respectively. To determine whether FGF2 is a direct target of miR-936, cells were plated into 24-well plates and co-transfected with wt or mut luciferase reporter plasmid and miR-936 mimics or miR-control using Lipofectamine™ 2000 according to the manufacturer’s protocol. After 48 h of transfection, cells were assayed using a Dual-Luciferase Reporter Assay system (Promega Corporation, Madison, WI, USA). The firefly luciferase activity was normalized to Renilla luciferase activity.

Western blot analysis

Tissue samples or cells were lysed in cold radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China). The concentration of isolated total protein was quantified using a Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer’s protocol. Equal amounts of protein were loaded per lane and run on a 10% SDS-PAGE gel, transferred to PVDF membranes, and then blocked for 2 h in 5% powdered skim milk diluted in Tris-buffered saline containing 0.1% Tween® 20 (TBST) at room temperature. After incubating with primary antibodies overnight at 4 °C, the membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit (cat. no. ab205718) or goat anti-mouse (cat. no. ab205719) secondary antibody (1:4,000 dilution; Abcam, Cambridge, UK). Protein bands were visualized using an enhanced chemiluminescence reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The following primary antibodies were used: rabbit anti-human FGF2 antibody (cat. no. ab208687; Abcam), rabbit anti-human p-PI3K (cat. no. ab182651; Abcam), rabbit anti-human PI3K (cat. no. ab191606; Abcam), mouse anti-human p-Akt (cat. no. sc-514032; Santa Cruz Biotechnology, CA, USA), mouse anti-human Akt (cat. no.sc-81434; Santa Cruz Biotechnology), and mouse anti-human GAPDH antibody (cat. no. ab125247; Abcam). All primary antibodies were used at 1:1,000 dilution.

Immunohistochemical (IHC) analysis

After baking at 60 °C for 2  h, 0.5 mm tumor sections of xenografts were sliced, and dried, deparaffinized, followed by rehydrating in xylene and graded ethanol for 10 min. Next, antigen retrieval was achieved via heating (100 °C) for 25 min in citrate buffer. After incubation with primary antibody against Ki-67 (cat. no. ab92742; Abcam) at 4 °C overnight, all sections were probed with the secondary antibody at room temperature for 1 h. after being rinsed with PBS, all sections were added with diaminobenzidine. The hematoxylin, dehydrated, mounted with VectaMount (Vector Laboratories) were applied to counter stain the sections. The slides were observed under a light microscope (Olympus Corporation).

Statistical analysis

Each assay was repeated thrice, and all results were presented as the mean ± standard deviation. Chi-squared test was performed to determine the correlation between miR-936 and the clinicopathological characteristics of EOC patients. Student’s t-test was used for comparing two groups. One-way analysis of variance, followed by Student-Newman-Keuls post-hoc tests, was used to analyze the differences between multiple group comparisons. For non-parametric data, a Mann Witnney Test was used for evaluating differences between two groups, while the comparisons among more than two groups were conducted using a Kruskall Wallis Test. The correlation between the miR-936 and FGF2 mRNA levels in tissue specimens was evaluated using Spearman’s correlation analysis. P<0.05 was considered statistically significant.

Results

MiR-936 is downregulated in EOC and is correlated with several clinicopathological factors in EOC patients

To investigate the clinical significance of miR-936 in EOC, 51 pairs of EOC tissues and adjacent normal tissues were collected and analyzed by RT-qPCR. Results showed that miR-936 was significantly downregulated in EOC tissues compared to adjacent normal tissues (Figure 1A, P<0.05). Next, we determined whether miR-936 expression is correlated with the clinicopathological factors of EOC patients. As shown in Table 1, the downregulation of miR-936 levels was significantly associated with tumor size (P=0.035), FIGO stage (P=0.016), and lymphatic metastasis (P=0.017) in EOC patients. In addition, miR-936 expression was measured in several EOC cell lines, including OVCAR3, SKOV3, CAOV-3, and ES-2 cells. The normal human ovarian epithelial cell line NOEC was utilized as normal control. Compared to NOEC, all four EOC cell lines expressed lower levels of miR-936 (Figure 1B, P<0.05). Thus, the findings suggested that miR-936 downregulation is closely related to EOC progression and development.

Figure 1

MiR-936 is downregulated in EOC tissues and cell lines. (A) MiR-936 expression was measured in 51 pairs of EOC tissues and adjacent normal tissues by RT-qPCR. *P<0.05 vs normal. (B) MiR-936 expression levels were measured between EOC cell lines (OVCAR3, SKOV3, CAOV-3, and ES-2) and a normal human ovarian epithelial cell line (NOEC) by RT-qPCR. *P<0.05 versus NOEC.

Table 1

Association of miR-936 expression with clinicopathological factors of EOC patients

Factors	miR-936 expression		P
Low	High
Age at diagnosis (years)			0.657
<60	12	10
≥60	14	15
Tumor size (cm)			0.035a
<2	10	17
≥2	16	8
Differentiated degree			0.202
G1	7	11
G2+G3	19	14
FIGO stage			0.016a
I-II	10	18
III-IV	16	7
Lymphatic metastasis			0.017a
No	8	16
Yes	18	9

Notes: aP<0.05.

Abbreviations: G1, Well differentiated; G2, Moderately differentiated; G3, Poorly differentiated.

MiR-936 suppresses the malignant biological behaviors of EOC cells in vitro

Considering that miR-936 is downregulated in EOC patients and cell lines, we aimed to determine the role of miR-936 in the aggressive of EOC cells. The SKOV3 and CAOV-3 cell lines, which showed relatively lower miR-936 expression levels compared to the other EOC cell lines, were selected for subsequent functional experiments. The two cell lines were transfected with miR-936 mimics to overexpress miR-936 (Figure 2A, P<0.05) or with miR-control (negative control). CCK-8 assay and flow cytometry were conducted to examine the effects of miR-936 upregulation on EOC cell proliferation and apoptosis. Compared to cells in the miR-control group, SKOV3 and CAOV-3 cells overexpressing miR-936 showed reduced proliferation (Figure 2B, P<0.05) and increased cell apoptosis (Figure 2C, P<0.05). Furthermore, the migratory (Figure 2D, P<0.05) and invasive (Figure 2E, P<0.05) abilities of SKOV3 and CAOV-3 cells were significantly suppressed following miR-936 upregulation. The above results implied that miR-936 plays a tumor suppressive role in EOC progression.

Figure 2

MiR-936 inhibits SKOV3 and CAOV-3 cell proliferation, migration, and invasion but promotes cell apoptosis. (A) SKOV3 and CAOV-3 cells were transfected with miR-936 mimics or miR-control and were analyzed after 48 h of transfection. MiR-936 levels were measured by RT-qPCR. *P<0.05 vs miR-control. (B and C) CCK-8 and flow cytometry assays were conducted to investigate cell proliferation and apoptosis in miR-936-overexpressing SKOV3 and CAOV-3 cells. Ectopic miR-936 expression notably decreased the proliferation and promoted the apoptosis of SKOV3 and CAOV-3 cells. *P<0.05 vs miR-control. (D and E) Migration and invasion assays were conducted to investigate the migratory and invasive abilities of miR-936 overexpression SKOV3 and CAOV-3 cells. The number of migrated and invaded SKOV3 and CAOV-3 cells was obviously lower in miR-936 mimics group than those in miR-control group. *P<0.05 vs miR-control.

FGF2 is a direct target of miR-936 in EOC cells

To investigate the mechanism underlying the effects of miR-936 in EOC, we searched three public databases for potential miR-936 targets. FGF2, which contains a complementary site for miR-936 (Figure 3A), was previously reported to be involved in the occurrence and development of EOC.20–23 We conducted a luciferase reporter assay to verify whether FGF2 is a direct target of miR-936 in EOC cells. Luciferase reporter plasmids carrying wild-type (wt) FGF2 3ʹ-UTR and mutated (mut) FGF2 3ʹ-UTR were generated and co-transfected with miR-936 mimics or miR-control into SKOV3 and CAOV-3 cells. The results revealed that ectopic miR-936 expression reduced the luciferase activity of SKOV3 and CAOV-3 cells transfected with the plasmid harboring wt FGF2 3ʹ-UTR (Figure 3B, P < 0.05). However, miR-936 overexpression did not affect the luciferase activity of the plasmid carrying mut FGF2 3ʹ-UTR.

Figure 3

FGF2 is a direct target of miR-936 in EOC cells. (A) Schematic of the wild-type (wt) and mutant (mut) binding sites of miR-936 in the FGF2 3ʹ-UTR. (B) Luciferase activity was measured at 48 h after transfection in SKOV3 and CAOV-3 cells co-transfected with wt or mut luciferase reporter plasmid and miR-936 mimics or miR-control. *P<0.05 vs miR-control. (C) FGF2 mRNA levels in EOC tissues was measured by RT-qPCR relative to adjacent normal tissues. *P<0.05 vs normal. (D) Correlation between miR-936 and FGF2 mRNA levels in EOC tissues was determined by Spearman’s correlation analysis. r = −0.5162, P=0.0001. (E and F) Following miR-936 overexpression in SKOV3 and CAOV-3 cells, FGF2 mRNA and protein levels were measured by RT-qPCR and Western blot analysis, respectively. *P<0.05 vs miR-control.

FGF2 expression levels were measured in EOC tissues and adjacent normal tissues by RT-qPCR. FGF2 mRNA levels were found to be higher in EOC tissues than in adjacent non-tumor tissues (Figure 3C, P < 0.05). Furthermore, miR-936 and FGF2 mRNA levels were inversely correlated in EOC tissues (Figure 3D; r=−0.5162, P=0.0001). Moreover, restoration of miR-936 expression evidently downregulated FGF2 expression in SKOV3 and CAOV-3 cells at both the mRNA (Figure 3E, P<0.05) and protein (Figure 3F, P<0.05) levels. These results provided strong evidence that FGF2 is a direct target of miR-936 in EOC cells.

FGF2 knockdown has similar inhibitory effects as mir-936 overexpression in EOC cells

To determine the functional role of FGF2 in the development of EOC, we performed knockdown experiments with FGF2 siRNA (si-FGF2) or negative control siRNA (si-NC) in SKOV3 and CAOV-3 cells. FGF2 protein knockdown in si-FGF2 transfected SKOV3 and CAOV-3 ells was confirmed by Western blot analysis (Figure 4A, P < 0.05). Next, we investigated the role of FGF2 knockdown in proliferation, apoptosis, migration, and invasion of EOC cells by conducting CCK-8, flow cytometry, and migration and invasion assays, respectively. Depletion of FGF2 inhibited cell proliferation (Figure 4B, P < 0.05), induced cell apoptosis (Figure 4C, P < 0.05), and inhibited cell migration (Figure 4D, P < 0.05) and invasion (Figure 4E, P < 0.05) in SKOV3 and CAOV-3 cells. Accordingly, the phenotypes of FGF2 knockdown in EOC cells were similar to those induced by miR-936 overexpression. Taken together, the above findings provided further evidence that FGF2 is a functional downstream mediator of miR-936 in EOC cells.

Figure 4

Effects of FGF2 knockdown on the malignant behaviors of SKOV3 and CAOV-3 cells in vitro. (A) Protein expression of FGF2 in SKOV3 and CAOV-3 cells after transfection with si-FGF2 or si-NC were measured by Western blot analysis. *P<0.05 vs si-NC. (B–E) Cell proliferation, apoptosis, and migration and invasion were determined by CCK-8, flow cytometry, and migration and invasion assays, respectively, after si-FGF2 or si-NC transfection in SKOV3 and CAOV-3 cells. *P<0.05 versus si-NC.

FGF2 restoration alleviates miR-936-mediated inhibition of the malignant behavior in EOC cells

Based on the previous results, we hypothesized that FGF2 downregulation is essential for the miR-936-mediated suppression of malignant phenotypes in EOC cells. To confirm this hypothesis, miR-936-overexpression SKOV3 and CAOV-3 cells were further transfected with FGF2-overexpression plasmid (pcDNA3.1-FGF2) or empty plasmid (pcDNA3.1) as a negative control. TFGF2 protein levels were recovered in miR-936 mimics-transfected SKOV3 and CAOV-3 cells after co-transfection with pcDNA3.1-FGF2 (Figure 5A, P < 0.05). Functional assays revealed that miR-936-induced inhibition of SKOV3 and CAOV-3 cell proliferation (Figure 5B, P < 0.05), promotion of cell apoptosis (Figure 5C, P < 0.05), and suppression of cell migration (Figure 5D, P < 0.05) and invasion (Figure 5E, P < 0.05) were partly abrogated by FGF2 overexpression. These results demonstrated that miR-936 inhibited the malignant biological behaviors of EOC cells, at least partly, by downregulating FGF2 expression.

Figure 5

Rescuing FGF2 levels abolishes miR-936-mediated suppression of malignant phenotypes of SKOV3 and CAOV-3 cells. (A) SKOV3 and CAOV-3 cells were co-transfected with miR-936 mimics and pcDNA3.1-FGF2 or pcDNA3.1. After 72 h of transfection, FGF2 protein levels measured by Western blot analysis. *P<0.05 vs miR-control. **P<0.05 vs miR-936 mimics + pcDNA3.1. (B–E) CCK-8, flow cytometry, and migration and invasion assays were conducted to evaluate the proliferation, apoptosis, and migration and invasion of SKOV3 and CAOV-3 cells treated as described above. *P<0.05 vs miR-control. **P<0.05 vs miR-936 mimics + pcDNA3.1.

MiR-936 inhibits the activation of the PI3K/Akt pathway in EOC cells

Several studies have established that FGF2 is involved in the regulation of the PI3K/Akt pathway.21,24,25 We speculated that the PI3K/Akt signaling pathway in EOC cells could be deactivated by miR-936. To test this hypothesis, we measured the levels of proteins involved in the PI3K/Akt pathway in SKOV3 and CAOV-3 cells co-transfected with miR-936 mimics and pcDNA3.1-FGF2 or pcDNA3.1. Results of Western blot analysis indicated that p-PI3K and p-Akt protein levels were significantly downregulated in SKOV3 and CAOV-3 cells upon miR-936 upregulation. However, the total protein levels of PI3K and Akt were unaffected. In addition, the downregulation of p-PI3K and p-Akt levels in SKOV3 and CAOV-3 cells associated with miR-936 upregulation was reversed by FGF2 overexpression (Figure 6). These results suggested that miR-936-induced inhibition of FGF2 leads to the inactivation of the PI3K/Akt signaling pathway in EOC cells.

Figure 6

MiR-936 deactivates the PI3K/Akt pathway in EOC cells. SKOV3 and CAOV-3 cells were transfected with miR-936 mimics and pcDNA3.1-FGF2 or pcDNA3.1. After 72 h of transfection, the expression levels of proteins involved in the PI3K/Akt pathway were measured by Western blot analysis.

MiR-936 overexpression suppresses the growth of EOC in vivo

Given the inhibitory effect of miR-936 on proliferation of EOC cells in vitro, we used xenograft models in nude mice to determine whether miR-936 overexpression had a similar antitumor effect on tumor growth in vivo. Tumor xenograft volume (Figure 7A and B, P < 0.05) and weight (Figure 7C, P < 0.05) were significantly lower in the group treated with the miR-936 mimics than the miR-control group. In addition, the results of IHC analysis manifested that cells expressed Ki-67 in tumor xenografts of miR-936 mimics group was significantly less than that of miR-control group (Figure 7D), indicating that miR-936 overexpression could decrease EOC cell proliferation in vivo. Next, miR-936 expression was evaluated in the tumor xenografts using RT-qPCR. Compared to the miR-control group, miR-936 expression was significantly higher in tumor xenografts in the miR-936 mimics group (Figure 7E, P < 0.05). Furthermore, the protein levels of FGF2 and other important proteins associated with the PI3K/Akt pathway were measured in the tumor xenografts by Western blotting. Results revealed that the protein levels of FGF2, p-PI3K, and p-Akt were downregulated in tumor xenografts from the miR-936 mimics group (Figure 7F). Based on our findings, miR-936 overexpression inhibited tumor growth of EOC in vivo, which could be related to the downregulation of FGF2 and inactivation of the PI3K/Akt pathway.

Figure 7

MiR-936 inhibits tumor growth in vivo. (A) Representative images of tumor xenografts obtained from miR-936 mimics and miR-control groups. (B) Growth curves of tumor xenografts were measured every four days for four weeks. *P<0.05 vs miR-control. (C) Average tumor weight was recorded on the last day of the experiment. *P<0.05 vs miR-control. (D) The images showing IHC staining for Ki-67 expression. (E) MiR-936 expression levels in tumor xenografts were analyzed using RT-qPCR. *P<0.05 vs miR-control. (F) Western blot analysis was conducted to compare the protein levels of FGF2, p-PI3K, PI3K, p-Akt, and Akt in the tumor xenografts from miR-936 mimics- and miR-control-transfected cells.

Discussion

Accumulating evidence has revealed that a variety of miRNAs are aberrantly expressed in EOC.26–28 These dysregulated miRNAs play tumor-suppressing or oncogenic roles in EOC cells and participate in the pathological progression of EOC.29–31 Therefore, understanding the miRNAs that are associated with the malignant biological behavior of EOC are valuable for identifying potential therapeutic targets for the treatment of EOC patients. In the present study, we analyzed the expression status, clinical significance, and functions of miR-936 in EOC. In addition, we examined the potential molecular mechanism underlying the effects of miR-936 in EOC cells. Our study is the first to demonstrate a fundamental role for miR-936 in the progression and development of EOC.

MiR-936 is downregulated in non-small cell lung cancer tissues and cell lines, and miR-936 overexpression suppresses cell proliferation, induces cell cycle arrest, and impedes cell invasion in non-small cell lung cancer.17 MiR-936 expression is also downregulated in glioma tissues and cell lines. Glioma patients with lower miR-936 expression levels exhibit worse survival than patients with higher miR-936 expression.18 Functionally, miR-936 inhibits the growth of glioma cells both in vitro and in vivo.18 However, the expression and roles of miR-936 in EOC remain unclear. In this study, we performed RT-qPCR to analyze miR-936 expression in EOC tissues and cell lines. Results revealed that miR-936 expression is downregulated in EOC tissues and cell lines. In addition, low miR-936 expression was significantly correlated with the tumor size, FIGO stage, and lymphatic metastasis in EOC patients. Functional experiments demonstrated that restoration of miR-936 expression inhibited EOC cell proliferation, migration, and invasion in vitro; promoted cell apoptosis; and suppressed tumor growth in vivo. Therefore, miR-936 is a promising diagnostic biomarker and therapeutic target for EOC patients. However, in this study, we did not investigate whether miR-936 may affect the tumor metastasisi of EOC in vivo. It was a limitation of our current study, and we will resolve it in the near future.

E2F217 and CKS118 have been previously identified as direct targets of miR-936 in non-small cell lung cancer and glioma, respectively. However, the mechanisms underlying the regulatory roles of miR-936 in EOC progression remain unclear. Therefore, we predicted the putative miR-936 targets using bioinformatics analysis. The FGF2 gene contains a complementary site for miR-936. Luciferase reporter assays indicated that miR-936 can recognize and directly bind to the 3ʹ-UTR of FGF2. Furthermore, miR-936 and FGF2 expression levels were inversely correlated in EOC tissues. Consistent with the expected results, miR-936 overexpression significantly downregulated FGF2 mRNA and protein levels in EOC cells. FGF2 knockdown phenocopied the tumor suppressor activity of miR-936 overexpression. Rescuing FGF2 expression alleviated the miR-936-mediated inhibitory effects in EOC cells. Taken together, the above findings confirmed that FGF2 is a direct target gene of miR-936 in EOC cells.

FGF2, a member of the FGF family, is a prototypic growth factor.32 FGF2 is well characterized and possesses the typical features of the FGF family, which plays significant roles in various physiological and pathological processes.33 Previous studies reported that FGF2 is strongly expressed in various tumors, such as gastric cancer,34 breast cancer,35 glioma,36 and lung cancer.37 FGF2 is also highly expressed in EOC20 and has oncogenic activity in EOC formation and progression.21–23 Herein, we demonstrated that FGF2 expression is upregulated in EOC tissues and that FGF2 plays oncogenic roles that promote the oncogenicity of EOC cells. More importantly, we demonstrated that miR-936 targets FGF2 to inhibit the aggressive behaviors of EOC cells. Considering the key roles of FGF2 in EOC progression, FGF2 silencing via miR-936 upregulation could serve as an effective therapeutic strategy for the treatment of EOC patients.

Conclusion

In summary, the present study explored the biological functions of miR-936 in EOC progression. Our results revealed that miR-936 is frequently downregulated in EOC and is significantly correlated with the tumor size, FIGO stage, and lymphatic metastasis in EOC patients. MiR-936 exerts its tumor-suppressing roles in EOC, at least partially, by targeting the FGF2-mediated suppression of the PI3K/Akt pathway. Therefore, our findings could serve as the basis for the development of novel and effective therapeutic approaches for EOC patients.