MiR-27a promotes EMT in ovarian cancer through active Wnt/ 𝜷 -catenin signalling by targeting FOXO1

Abstract

BACKGROUND:

Ovarian cancer (OC) is the fifth most common type of cancer in women worldwide. MiR-27a plays an important role in the development of ovarian cancer. However, the exact function and molecular mechanism of miR-27a in epithelial-mesenchymal transition (EMT) has not been thoroughly elucidated to date.

METHODS:

Quantitative real-time PCR (qRT-PCR) was used to determine the expression of miR-27a and FOXO1 mRNA in ovarian tissues and cells. The function of miR-27a in ovarian cancer was investigated through overexpression and knockdown of miR-27a in vitro. Wound healing and Transwell assays were performed to evaluate the migration and invasive capacity of the cells. A luciferase reporter assay was conducted to confirm the interaction between miR-27a and FOXO1. Western blotting was used to evaluate FOXO1, EMT and Wnt/ $\beta$ -catenin relative protein expression.

RESULTS:

In our study, we found that the mRNA expression level of miR-27a was significantly higher in ovarian cancer tissues and in HO8910 and OV90 cells. Functional experiments showed that miR-27a overexpression potentiated the migration and invasion of HO8910 and OV90 cells, while miR-27a inhibition reduced the cells’ migration and invasion. Moreover, miR-27a upregulated the expression of mesenchymal cell markers and downregulated the expression of epithelial cell markers, which were restored via silencing of miR-27a expression. Subsequently, miR-27a was found to directly target and suppress the expression of FOXO1. Finally, we demonstrated that miR-27a promoted the progression of ovarian cancer cells and induced the process of EMT via the Wnt/ $\beta$ -catenin signalling pathway through inhibition of FOXO1.

CONCLUSIONS:

Taken together, these results indicate that targeting miR-27a and FOXO1 could represent a strategy for anticancer therapy in ovarian cancer.

Keywords

Ovarian cancer microRNA-27a FOXO1 EMT Wnt/β-catenin

1. Introduction

Ovarian cancer is the second leading cause of death among all gynaecological tumours and is a common female reproductive cancer; it ranks fifth among causes of death for female cancer patients [1, 2, 3]. In 2015, there were 22,500 deaths and 52,100 patients newly diagnosed with OC in China [4]. It is difficult to diagnosis this cancer in early stages due to a lack of significant signs or symptoms. The majority of OC cases are diagnosed at an advanced metastatic stage and have a poor outcome [5]. Thus, improving targeting therapies and studying the mechanisms underlying tumour metastasis are necessary for reducing OC mortality.

Epithelial-mesenchymal transition is defined as a dynamic process in which epithelial cells undergo multiple biochemical changes and assume a mesenchymal phenotype, which has motile and invasive characteristics [2]. Numerous studies have indicated that EMT is related to metastasis, which is defined as a process in which cancer cells spread from the primary tissue to surrounding tissues because the cells lose their cell-cell adhesion ability and gain migratory and invasive capabilities [1, 6, 7]. While undergoing the EMT process, cells upregulate their expression of mesenchymal cell markers (e.g., N-cadherin, vimentin, and fibronectin) and simultaneously downregulate epithelial markers (e.g., E-cadherin, $\alpha$ -catenin, and $\gamma$ -catenin) [2, 8]. Importantly, the aggressive properties of OC in terms of EMT is of major concern. It has been reported that TLR-mediated PI3K activation modulates the invasion and metastasis of ovarian cancer through EMT [5]. The expression and clinical role of EMT is related to factors in ovarian cancer.

MiRNAs are approximately 22-nucleotide single-stranded noncoding RNA molecules that are incorporated into the RNA-induced silencing complex (RISC) [9, 10, 11, 12]. Typically, the miRNAs play important roles in cell differentiation, proliferation, apoptosis and EMT. For instance, upregulation of miR-483-5p in human lung adenocarcinoma is correlated with the progression of the tumour via the activation of Wnt/ $\beta$ -catenin signalling to promote lung adenocarcinoma cell EMT [13]. Overexpression of miR-27 was found to increase EMT by activating the Wnt pathway during gastric cancer therapy [14]. Chae et al. demonstrated that upregulation of miR-27a may play a key role in lung cancer progression by regulating TGF- $\beta$ signalling [15]. Moreover, miR-27a can promote proliferation and metastasis of tumour cells by suppressing PHLPP2 and activating the AKT/GSK3 $\beta$ pathway during gastric cancer as a crucial oncogenic miRNA [16]. It has also been reported that miR-27a may be involved in the development of drug resistance, regulating the expression of MDR1/P-gp [17]. Interesting, high expression of miR-27a has been identified in a sub-group of patients with a very poor prognosis, which reveals that miR-27a is involved in ovarian cancer progression [18]. However, to the best of our knowledge, the role of miR-27a associated with EMT in OC metastasis and the molecular mechanisms involved have not previously been reported.

In the present study, we determined the regulatory role of miR-27a-induced EMT in ovarian cells. Wnt/ $\beta$ -catenin signalling plays an important regulatory role in the process of EMT [19, 20, 21, 22]; therefore, we focused on the Wnt/ $\beta$ -catenin signalling pathway. In this study, we found that overexpression of miR-27a in HO8910 and OV90 cells promoted the migration and invasion of the cells, and expression of E-cadherin proteins were decreased, while N-cadherin and vimentin increased following transfection with miR-27a. Moreover, we demonstrated that FOXO1 expression decreased in response to overexpression of miR-27a, and the mechanism behind this could be attributed to modulation of the Wnt/ $\beta$ -catenin signalling pathway. Taken together, our data suggest that upregulation of miR-27a may play a key role in promoting migration, invasion and activation of the Wnt/ $\beta$ -catenin signalling pathway by targeting FOXO1 in ovarian cells.

2. Methods and materials

2.1 Cell culture

OV90 (a high-grade serous ovarian cancer cell line) and HEK-29 (a human embryonic kidney epithelial cell line) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HOSEpiC (a human ovarian surface epithelial cell line) and HO8910 (an ovarian cancer cell line) cell lines were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were authenticated with short tandem repeat (STR) and mycoplasma analysis, which showed that the STR authentication was correct and that they were mycoplasma-negative. The cells were incubated in Dulbecco’s modified essential medium (DMEM; Gibco, Invitrogen) supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 $\mu$ g/ml), maintained at 37 ${}^{\circ}$ C in a humidified incubator containing 5% CO ${}_{2}$ , then stored in our laboratory and used for transfection.

2.2 Tumour tissue specimens

We obtained 37 paired ovarian cancer tissues and adjacent non-tumour tissues from patients who had undergone surgical resection at the Huizhou No. 2 Women’s and Children’s Healthcare Hospital. Patients who had preoperative chemotherapy or radiotherapy and patients who were diagnosed with other malignant diseases were excluded from our experimental group. Informed written consent was obtained from all participants, and ethical approval for the use of ovarian cancer specimens for this study was obtained from the Research Ethics Committee of Huizhou No. 2 Women’s and Children’s Healthcare Hospital.

2.3 Cell transfection

The miR-27a mimics and inhibitors (including mimic negative control: miR-NC; inhibitor negative control: anti-NC) and si-FOXO1 were chemically synthesized by Genepharma Corporation (Shanghai, China). OV90 and HOSEpiC cells were seeded in 6-well plates at 3.5 $\times$ 10 ${}^{5}$ cells/well and cultured for 12–18 h and then transfected with 100 pmol of the mimics, inhibitors of miR-27a or a negative control (NC) RNA using Lipofectamine 2000 and OPTI-MEM I reduced serum medium (Invitrogen Life, Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The sequences were as follows: miR-27a mimics, 5’-UUCACAGUGGCUAAGUUCCGC-3’, miR-27a inhibitor, 5’-UGGGGUAUUUGACAAACUGAC A-3’; si-FOXO1, sense: 5’-GAGGATAAGGGCGAGC AACGAGTACTGGTTGCTGTCGCCCTTATCCTTTTTT-3’, antisense: 5’-TAGAAAAAGGATAAGGGCG ACAGCAACCAGTACTCGTTGCTGTCGCCCTTATCC-3’. The transfection efficiency was measured 48 h later followed by qRT-PCR and western blot experiments.

2.4 Wound healing assays

Cells were seeded in 6-well plates to form a cell monolayer (near 90% confluence), and a wound was created by scratching the length of the well with a 10 $\mu$ L pipette tip. Then, the plate was washed three times with PBS to remove floating cells and photographed (time 0 h). The cells were incubated in low serum medium (2% FBS). Later, the wounded cultures were incubated in conditioned medium at 37 ${}^{\circ}$ C. At 0 and 1 day, images were captured using an inverted microscope to assess wound closure and then compared to determine differences in cell migration. Each assay was replicated 3 times.

2.5 Transwell invasion assay

The upper chamber of the Transwell was coated with Matrigel and then incubated at 37 ${}^{\circ}$ C for 1 h. After solidification, 1 $\times$ 10 ${}^{4}$ cells in 100 $\mu$ l of serum-free DMEM was added to the upper chambers, and 500 $\mu$ L of DMEM containing 10% FBS was added to the lower chamber. After incubation for 24 h, the cells on the upper surface of the membranes were removed with a cotton swab. The cells on the lower surface of the membranes were fixed with 4% paraformaldehyde for 10 min, washed three times with PBS, then stained with crystal violet for 10 min, washed with PBS, and subsequently air-dried. The cells that had invaded the lower surface of the membranes were counted in five different fields under a high-power microscope.

Table 1
The primer sets for qRT-PCR

Gene name	Primer sequence
FOXO1-F	5’-TGCATTTCGCTACCCGAGTT-3’
FOXO1-R	5’-GTGGCTGACAAGACTTAACTCAA-3’
miR-27a-F	5’-GCGCATTCACAGTGGCTAAG-3’
miR-27a-R	5’-CGGCCCAGTGTTCAGACTAC-3’;
N-Cadherin-F	5’-AGGGGACCTTTTCCTCAAGA-3’
N-Cadherin-R	5’-TCAAATGAAACCGGGCTATC-3’
E-cadherin-F	5’-TCACATCCTACACTGCCCAG-3’
E-cadherin-R	5’-AGTGTCCCTGTTCCAGTAGC-3’
Vimentin-F	5’-GGACCAGCTAACCAACGACA-3’
Vimentin-R	5’-AAGGTCAAGACGTGCCAGAG-3’
U6-F	5’-CTCGCTTCGGCAGCACA-3’
U6-R	5’-AACGCTTCACGAATTTGCGT-3’;
GAPDH-F	5’-AGCCCAAGATGCCCTTCAGT-3’
GAPDH-R	5’-CCGTGTTCCTACCCCCAATG-3’

cgcagcctgc agcccg.

2.6 RNA isolation and real-time PCR

Total RNA was prepared from tissues and cell lines using a Qiagen RNeasy kit per the manufacturer ’s instructions. The total RNA was treated with DNase I, and 1 $\mu$ g of total RNA was assayed using SYBR Premix Ex TaqTM (Takara) according to the manufacturer’s protocol and a stem-loop primer for miR-27a reverse transcription: 5’-GTCGTATCCAGTGCAGGGTC CGAGGTATTCGCACTGGATACGACGCGGAA-3’. All samples were carried out in triplicate on the same plate and U6 was utilized as the reference gene for miR-27a. Expression levels of other functional genes (vimentin, E-cadherin, N-cadherin, and FOXO1) were related to the expression level of GAPDH. Fold change in gene expression was calculated using the 2 ${}^{-\Delta\Delta\text{Ct}}$ method. Data are presented as fold change in gene expression normalized to U6 and relative to the mock-infected cells. The primer sequences are shown in Table 1.

Figure 1.

MiR-27a is upregulated in ovarian cancer tissues and cell lines. (A) Expression of miR-27a mRNA in ovarian normal tissues and ovarian cancer tissues. (B) The expression of miR-27a mRNA in HO8910 and OV90 cells compared with HOSEpiC cells. U6 was used as an internal reference. Bars represent means $\pm$ SD from three independent experiments. ** $p<$ 0.01, *** $p<$ 0.001.

2.7 Western blot analysis

Protein samples were resolved on 12% SDS-PAGE. The separated proteins were electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were blocked in blocking buffer (Tris-buffered saline containing 10% non-fat dry milk and 0.1% Tween 20) for 2 h. The membranes were probed with anti-N-cadherin (1:2000, Cell Signaling Technology, Boston, MA, USA), anti-E-cadherin (1:5000, BD Biosciences, San Jose, CA, USA), anti-vimentin (1:1,000, V6630, Sigma-Aldrich), anti-FOXO1 (1:1000, Cell Signaling Technology), and anti- $\beta$ -actin (1:3000, Cell Signaling Technology) antibodies. After washing, the membrane was incubated with the appropriate HRP-conjugated secondary antibodies, treated with ECL reagent and observed with ECL. The expression of $\beta$ -actin was detected with an anti- $\beta$ -actin mouse monoclonal antibody to demonstrate equal protein sample loading. The proteins were detected using a film-free gel imaging system (China, Clinx Science Instruments, cat. no. ClinxChemiScope 6000), and the gradation analysis used ImageJ software (National Institutes of Health, USA).

2.8 Targeting prediction analysis

Putative binding genes of miR-27a were analysed by the online bioinformatic tool Targetscan (http://www. targetscan.org) and starbase v2.0 (http://starbase.sysu. edu.cn).

2.9 Plasmid construction

The luciferase reporter plasmids pGL3-WT-FOXO1 and pGL3-MUT-FOXO1, containing a predicted wild-type or mutant miR-27a binding site, were constructed by PCR amplification from genomic DNA of ovarian cancer cells. Then, the fragment was cloned into the pGL3 basic vector (Promega), which contains a modified coding region of firefly (Photinus pyralis) luciferase. The pRL-TK plasmid (Promega), containing the Renilla luciferase reporter, served as an internal control.

2.10 Luciferase reporter assay

To determine the role of miR-27a in FOXO1 3’-UTR promoter activity, 293T cells were pre-cultured on 12-well plates and transfected with 1 $\mu$ g miR-27a, 0.1 $\mu$ g pRL-TK for normalization and 1 $\mu$ g pGL3-WT-FOXO1 or pGL3-MUT-FOXO1. Finally, cells were lysed in passive lysis buffer (Promega) and their luciferase activities were measured with a dual-luciferase assay system (Promega) according to the manufacturer’s instructions. The results are presented as fold-change of relative luciferase activities compared with the mock-treated group transfected with empty vectors. All assays were repeated at least three times with each experiment performed in triplicate.

2.11 Statistical analysis

All experiments were independently repeated three times. All values in the text and figures are expressed as the mean $\pm$ SD of these observations. Student’s t-test and one-way analysis was used for raw data analysis. Data analyses were conducted using SPSS 20.0 (IBM Corporation, Armonk, NY, USA) statistical software and GraphPad Prism 6.0 (GraphPad, La Jolla, CA, USA) programme. A $p$ -value of $<$ 0.05 for all statistical analyses was set to indicate significant differences.

Figure 2.

Overexpression of miR-27a promotes ovarian cancer cell EMT, cell motility, and invasion. (A) qRT-PCR was used to detect the mRNA expression level of miR-27a in HO8910 and OV90 cells. U6 was used as an internal reference. (B) HO8910 and OV90 cells were MOCK-transfected (control) or transfected with miR-27a mimics, miR-NC, and then their migration ability was measured by a wound healing assay. (C and D) The invasion assays of HO8910 and OV90 cells transfected with miR-27a mimics, NC, or controls were performed by using Transwell assays. Magnification, $\times$ 200 ** $p<$ 0.01. (E) Microscopic views of cells transfected with miR-27a mimics, miR-NC or controls (magnification, $\times$ 200, scale bars, 50 $\mu$ m). (F) Quantitative RT-PCR of E-cadherin, N-cadherin and vimentin in the HO8910 and OV90 cells, which were MOCK-transfected (control) or transfected with miR-27a mimics, miR-NC, with GAPDH used as the quantitative control. More than three independent experiments were performed in triplicate (two-sided independent Student’s t test, * $p<$ 0.05, ** $p<$ 0.01). (G) Western blot analysis of E-cadherin, N-cadherin and vimentin in the HO8910 and OV90 cells, which were transfected with miR-27a mimics, miR-NC or controls, with $\beta$ -actin as the quantitative control.

Figure 3.

Silencing miR-27a inhibited ovarian cancer cell EMT, cell motility, and invasion. (A) Knockout of miR-27a in HO8910 and OV90 cells followed by measuring the mRNA expression level of miR-27a detected by qRT-PCR. (B) Scratch migration and (C and D) Transwell analyses were used to determine the effect of silencing of miR-27a on the migratory and invasive capability of HO8910 and OV90 cells. * $p<$ 0.05, ** $p<$ 0.01 compared with the negative control group (anti-NC). (E) Microscopic views of cells treated with or without miR-27a inhibitors (magnification, $\times$ 200, scale bars, 50 $\mu$ m). (F and G) The expression levels of E-cadherin, N-cadherin and vimentin in the HO8910 and OV90 cells with knockout of miR-27a were determined by qRT-PCR (F) and WB (G).

3. Results

3.1 MiR-27a is upregulated in ovarian cancer tissues and cell lines

Numerous studies have shown that high expression of miR-27a is associated with cancer cell proliferation, migration and invasion [14, 17, 23, 24]. The expression level of miR-27a was tested by qRT-PCR. Figure 1A shows that the expression level of miR-27a is dramatically higher in ovarian cancer tissues compared to the corresponding normal tissues. Furthermore, we compared the expression of miR-27a in ovarian cancer cell lines (HO8910 and OV90) with human ovarian surface epithelial (HOSEpiC) cells. We found a 2.6- and 4.5-fold increased expression level of miR-27a in the ovarian cancer cell lines HO8910 and OV90, respectively (Fig. 1B).

3.2 MiR-27a promotes the migration and invasive capacity of ovarian cancer cells, and induces EMT activation

To explore the role of miR-27a in ovarian cancer cells, first, the HO8910 and OV90 cells were transfected with miR-27a mimics. The results showed that pretreatment with miR-27a mimics led to a 3.8- and 4.1-fold increased concentration of miR-27a, while there was no significant difference between the control group and the NC group (Fig. 2A). Furthermore, the wound-healing capacity of HO8910 and OV90 cells, which were transfected with miR-27a mimics, was dramatically greater than that of HO8910 and OV90 cells treated with NC (Fig. 2B and Supplement Fig. S1).

We next determined the effect of miR-27a on ovarian cancer cell invasion. Transwell invasion assays showed that the invasive ability of HO8910 and OV90 cells was significantly increased when they overexpressed miR-27a compared with the control group (Fig. 2C and D). As shown in Fig. 2E, miR-27a overexpression resulted in a dramatic transformation towards a mesenchymal cell phenotype, especially in HO8910 cells, which showed an altered cell morphology into a long spindle shape.

To obtain further insights into the EMT-promoting capability of miR-27a in ovarian cancer, E-cadherin, N-cadherin and vimentin were detected using qRT-PCR and western blotting. The results revealed that miR-27a overexpression might significantly increase both the mRNA level and protein level of N-cadherin and vimentin but it decreased E-cadherin compared with the NC cells (Fig. 2F and G). There was no significant difference between the control group and the NC group as shown in Fig. 2A–G. Generally, we could conclude that miR-27a promoted the migration and invasion of ovarian cancer cells. Moreover, their capacity of EMT was obviously increased while overexpressing miR-27a.

3.3 Knockdown of miR-27a inhibited cell motility, invasion and EMT of ovarian cancer cells

To further test the function of miR-27a in ovarian cancer cells, an miR-27 inhibitor was transfected into HO8910 and OV90 cells. It resulted in decreases in miR-27a expression by 67% and 79% in HO8910 and OV90 cells, respectively, compared with the expression of miR-27a in the miR-NC group (Fig. 3A). Under the effect of the miR-27a inhibitor, we found that their migration ability was decreased after inhibitor treatment (Fig. 3B and Supplement Fig. S2). As we had supposed, the number of cells invading through the membrane was also significantly decreased compared with the control group (Fig. 3C and D). Knockdown of miR-27a resulted in a significant change in morphology of the OV90 cells, while no significant change was observed for HO8910 (Fig. 3E). To examine the effect of miR-27a on the EMT, the expression of E-cad, N-cad and vimentin were assessed. Consistent with our expectation, the expression of these two genes (N-cad and vimentin) were decreased and only E-cad expression was enhanced following miR-27a inhibitor treatment in both HO8910 and OV90 cells (Fig. 3F). In addition, the western blot results showed decreased expression of N-cad and vimentin proteins in both HO8910 and OV90 cells, and meanwhile the protein level of E-cad was increased (Fig. 3G). Taken together, our findings indicate the miR-27a inhibitor could block the capacity for migration, invasion and EMT in ovarian cancer cells.

Figure 4.

MiR-27a directly targets the FOXO1 3’-UTR. (A) Binding sites of miR-27a in the 3’-UTR of FOXO1 are shown. (B) The relative luciferase activity was measured in HEK293 cells co-transfected with the WT or MUT FOXO1 3’-UTR with miR-27a mimics or miR-NC. (C and D) The relative mRNA and protein levels of FOXO1 were detected in cells transfected with or without miR-27a mimics and miR-27a inhibitors. (E and F) The expression level of FOXO1 in the tumour and normal adjacent tissues were determined by qRT-PCR (E) and WB (F). * $p<$ 0.05, ** $p<$ 0.01 vs controls.

Figure 5.

MiR-27a activates the Wnt/ $\beta$ -catenin pathway through FOXO1 to exert its biologic effects on ovarian cancer cells. (A and B) Knockout of FOXO1 in HO8910 and OV90 cells by shRNA followed by the mRNA expression level of FOXO1 being detected by qRT-PCR (A) and the protein level of FOXO1 being detected by western blotting (B). (C) Wound healing assay and (D) Transwell assay of HO8910 and OV90 cells for the miR-27 inhibitor group, the si-FOXO1 group, and the miR-27 inhibitor in combination with si-FOXO1. * $p<$ 0.05, ** $p<$ 0.01. (E) Western blot analysis of FOXO1, E-cadherin, N-cadherin, and vimentin in the indicated cells, with $\beta$ -actin as the loading control. (F) Western blot analysis detected the expression levels of $\beta$ -catenin and c-myc in HO8910 cells treated with anti-NC, miR-27a inhibitors, miR-27a inhibitors plus si-FOXO1, and si-FOXO1. * $p<$ 0.05, ** $p<$ 0.01.

3.4 FOXO1 is a functional target of miR-27a in both HO8910 and OV90 cells

To dissect the molecular mechanism involved in miR-27a regulated metastasis in ovarian cancer cells, the putative binding proteins of miR-27a were analysed by the online bioinformatic tool RNA hybrid. The results showed that the 3’-UTR of FOXO1 carried a binding sequence for miR-27a (Fig. 4A). Then, we constructed luciferase reporters for the FOXO1 gene that contained either a wild-type (WT) 3’-UTR or a mutant (mut) 3’-UTR with mutated sequences in the miR-27a binding site. Cells were cotransfected with miR-27a mimics together with the plasmids pGL3-WT-FOXO1 or pGL3-mut-FOXO1. The FOXO1 3’-UTR activity was measured with a luciferase assay. The results revealed that the wild-type FOXO1 reporter gene (pGL3-WT-FOXO1) activity was clearly suppressed by miR-27a overexpression compared with the miR-NC group. In contrast, the mutant FOXO1 reporter gene (pGL3-mut-FOXO1) was not significantly affecting by miR-27a transfection (Fig. 4B).

We also determined whether miR-27a is involved in regulation of the expression of FOXO1. As shown in Fig. 4C and D, overexpression of miR-27a led to a significantly decreased expression level of FOXO1 mRNA and protein, but their expression increased substantially in cells that were treated with miR-27a inhibitors. The findings for HO8910 cells and OV90 cells were concordant. Moreover, qRT-PCR and western blot analysis shown that the expression level of FOXO1 was decreased in ovarian cancer tissues compared with normal tissues (Fig. 4E and F). Taken together, miR-27a directly targets FOXO1 and inhibits its expression in ovarian cancer cell lines.

3.5 MiR-27a activates the Wnt/ $\beta$ -catenin pathway by targeting FOXO1

qRT-PCR and western blotting were performed to investigate whether using si-FOXO1 could inhibit the mRNA and protein level of FOXO1 (Fig. 5A and B). Knockdown of FOXO1 caused a significant increase in cell migration and invasive capacity (Fig. 5C and D). Co-transfection with si-FOXO1 and miR-27a inhibitors restored the effect of miR-27a and the cells’ capacity for cell migration and invasion, compared with cells only treated with miR-27a inhibitors. Similarly, western blot assays showed that knockdown of FOXO1 could decrease the expression of E-cadherin while increasing the levels of N-cadherin and vimentin. Meanwhile, co-treatment with si-FOXO1 and the miR-27a inhibitor led to downregulation of E-cadherin and upregulation of N-cadherin and vimentin, which is completely opposite to the results in the miR-27a inhibitor treatment group (Fig. 5E). It is known that activation of Wnt/ $\beta$ -catenin signalling in cancer often drives a transcriptional programme, such as transcriptional repression of E-cadherin, that is reminiscent of an EMT, which can affect gene expression and cell migration. $\beta$ -catenin and c-Myc are of great importance in the Wnt/ $\beta$ -catenin signalling pathway [13, 24]. Moreover, we found that silencing FOXO1 could increase the expression level of $\beta$ -catenin and c-Myc, but they were decreased in the miR-27a inhibitor treated cells. However, the concentrations of $\beta$ -catenin and c-Myc were remarkably higher after dual treatment compared with miR-27a inhibitor individual treatment (Fig. 5F). Thus, knocking down FOXO1 partially reversed the effect of miR-27a inhibitors on HO8910 cells. These results have confirmed that miR-27a promoted EMT partly through active Wnt/ $\beta$ -catenin signalling by targeting FOXO1 in ovarian cancer cells.

4. Discussion

Ovarian cancer has become one of the significant issues in recent years. Approximately 6,700 women in the UK are diagnosed with ovarian cancer each year. Therefore, it is necessary to explore the molecular mechanisms underlying ovarian cancer. Many studies have shown that miRNA has an important role in cellular homeostasis by regulating cellular signalling in such processes as proliferation, migration, and invasion, and breakdown of this management can finally lead to carcinogenesis [13, 25, 26, 27]. A growing body of evidence suggests that miR-27a exerts important roles in many cancers. For instance, miR-27a negatively regulates SMAD2 and SMAD4 by binding to the 3’-UTR of SMAD2 and SMAD4 and impairs their functions in lung cancer cells [16]. MiR-27a is highly expressed in GC tissues and cells, and it might promote cell proliferation, migration and invasion by targeting SF-RP1 via activation of the Wnt/ $\beta$ -catenin signalling pathway [24]. Previous studies have demonstrated that miR-27a is associated with a poor prognosis and aggressive behaviour in epithelial ovarian cancer tissues [23]. In our study, we found that miR-27a expression was significantly higher in ovarian cancer tissues and cell lines, and it promoted ovarian cancer cell migration and invasion.

EMT plays an important role in cancer metastasis. EMT occurs under physiological conditions; however, is also a key mechanism required for malignant progression. “Oncogenic EMT” allows tumour cells to acquire invasive properties and to develop metastatic growth characteristics. Moreover, it protects them from hostile conditions during the dissemination process [28]. In this study, we found that miR-27 might be responsible for promoting migration, invasion and EMT, while miR-27a inhibition markedly weakened EMT and the invasive capacity of ovarian cancer cells. All of these findings indicate that miR-27a plays an oncogenic role in OC by potentiating the metastatic behaviours and EMT of OC cells.

FOXO1, belonging to the O subgroup of forkhead transcription factors, is a well-known tumour suppressor and has been found to play an important role in the development and progression of various human cancers [29, 30, 31, 32, 33]. FOXO1 is known to be associated with cancer cell migration, invasion and EMT in vitro as well as tumourigenicity and metastasis in vivo [9, 29, 30, 34, 35]. This study showed that miR-27a targeting the expression of FOXO1. Overexpression of miR-27a significantly decreased the mRNA level of FOXO1. Importantly, knockout of FOXO1 promote the migration, invasion, and EMT of cancer cells, as well as overexpression of miR-27a. These findings indicate that miR-27a could modulate activation of EMT by inhibiting FOXO1. This effect has been supported by previous studies, such as reports that adipose tissue-secreted miR-27a promotes liver cancer by targeting FOXO1 in obese individuals. Chitosan degradation products promote nerve regeneration by stimulating Schwann cell proliferation via the miR-27a/FOXO1 axis [36]. Furthermore, our results showed that si-FOXO1 upregulated the expression of $\beta$ -catenin and c-myc, whereas miR-27a inhibitors decreased the expression of $\beta$ -catenin and c-myc. These findings revealed that miR-27a regulated EMT via Wnt/ $\beta$ -catenin signalling pathway stimulation by targeting FOXO1.

As an important regulator of EMT, the Wnt signalling pathway plays a crucial role [37, 38, 39]. Several signalling pathways, including Wnt/ $\beta$ -catenin, notch, and PI3K/Akt, are aberrantly activated and function in the progression of ovarian cancer [5]. Inhibitory effects of salinomycin on invasive abilities is mediated by repressing the EMT programme via inhibition of the Wnt/ $\beta$ -catenin pathway [8]. Meanwhile, $\beta$ -catenin and c-Myc are of great importance in the Wnt/ $\beta$ -catenin signalling pathway [13]. Several studies have reported that FOXO1 activation in various cell lines can inhibit cell survival, which can be attributed to modulation of Wnt/ $\beta$ -catenin signalling [30, 34, 40]. Our results established FOXO1, a negative regulator of Wnt $\beta$ -catenin signalling, as a direct functional effector of miR-27a that increases the migratory, invasive ability and EMT of OC cells.

In conclusion, the present study showed that miR-27a upregulates the migration, invasion and EMT of ovarian cancer cells. Moreover, miR-27a has an important role in increasing the expression of Wnt/ $\beta$ -catenin signalling related proteins in ovarian cancer cells. Meanwhile, overexpression of miR-27a remarkably inhibits the expression of FOXO1. Thus, our studies have advanced the understanding of the molecular mechanism of miR-27a in the regulation of EMT activation and the Wnt/ $\beta$ -catenin signalling pathway. Inhibition of miR-27a or restoration of FOXO1 expression can potentially serve as an effective treatment for ovarian cancer. Nevertheless, due to the complex nature of this multi-factorial disease, further studies are still required to unravel its specific regulation and aetiology.

Supplementary data

The supplementary files are available to download from https://dx-doi-org.web.bisu.edu.cn/10.3233/CBM-181229.

sj-docx-1-cbm-10.3233_CBM-181229.docx - Supplemental material

Supplemental material, sj-docx-1-cbm-10.3233_CBM-181229.docx

Footnotes

Acknowledgments

We would like to give our sincere gratitude to the reviewers for their constructive comments.

Conflict of interest

The authors declare that they have no conflict of interest.

Abbreviations

References

Koo

et al., CXCL11 mediates TWIST1-induced angiogenesis in epithelial ovarian cancer, Tumor Biol 39 (2017), 568835074.

Solheim

et al., Epithelial-mesenchymal transition markers in malignant ovarian germ cell tumors, Apmis 125 (2017), 781–786.

Chebouti

et al., EMT-like circulating tumor cells in ovarian cancer patients are enriched by platinum-based chemotherapy, Oncotarget (2017).

Samardzija

et al., Knockdown of stem cell regulator Oct4A in ovarian cancer reveals cellular reprogramming associated with key regulators of cytoskeleton-extracellular matrix remodelling, Sci Rep 7 (2017), 46312.

Park

G.B.

Chung

Y.H.

and Kim

, Induction of galectin-1 by TLR-dependent PI3K activation enhances epithelial-mesenchymal transition of metastatic ovarian cancer cells, Oncol Rep 37 (2017), 3137–3145.

et al., Salinomycin repressed the epithelial-mesenchymal transition of epithelial ovarian cancer cells via downregulating Wnt/β-catenin pathway, Onco Targets Ther 10 (2017), 1317–1325.

et al., MicroRNA-424 inhibits cell migration, invasion, and epithelial mesenchymal transition by downregulating doublecortin-like kinase 1 in ovarian clear cell carcinoma, Int J Biochem Cell Biol 85 (2017), 66–74.

Sun

et al., GOLPH3 induces epithelial-mesenchymal transition via Wnt/beta-catenin signaling pathway in epithelial ovarian cancer, Cancer Med 6 (2017), 834–844.

Zang

et al., miR-215 promotes cell migration and invasion of gastric cancer cell lines by targeting FOXO1, Neoplasma 64 (2017), 579–587.

10.

Liu

D.Z.

et al., MicroRNA-9 promotes the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1, Clin Transl Oncol 19 (2017), 1133–1140.

11.

Yin

et al., MiR-615-3p inhibits the osteogenic differentiation of human lumbar ligamentum flavum cells via suppression of osteogenic regulators GDF5 and FOXO1, Cell Biol Int 41 (2017), 779–786.

12.

Luo

et al., MicroRNA-21 regulates hepatic glucose metabolism by targeting FOXO1, Gene 627 (2017), 194–201.

13.

Song

et al., miR-483-5p Promotes Invasion and Metastasis of Lung Adenocarcinoma by Targeting RhoGDI1 and ALCAM, Cancer Res 74 (2014), 3031–3042.

14.

Zhang

et al., miR-27 promotes human gastric cancer cell metastasis by inducing epithelial-to-mesenchymal transition, Cancer Genet-Ny 204 (2011), 486–491.

15.

Chae

D.K.

et al., MIR-27a regulates the TGF-beta signaling pathway by targeting SMAD2 and SMAD4 in lung cancer, Mol Carcinog 56 (2017), 1992–1998.

16.

Zuccarini

et al., Modulation of the TGF-beta1-induced epithelial to mesenchymal transition (EMT) mediated by P1 and P2 purine receptors in MDCK cells, Purinergic Signal 13 (2017), 429–442.

17.

et al., MiR-27a modulates MDR1/P-glycoprotein expression by targeting HIPK2 in human ovarian cancer cells, Gynecol Oncol 119 (2010), 125–130.

18.

Eitan

et al., Tumor microRNA expression patterns associated with resistance to platinum based chemotherapy and survival in ovarian cancer patients, Gynecol Oncol 114 (2009), 253–259.

19.

Liu

et al., Tiam1 promotes thyroid carcinoma metastasis by modulating EMT via Wnt/beta-catenin signaling, Exp Cell Res 362 (2018), 532–540.

20.

Duan

et al., TET1 inhibits EMT of ovarian cancer cells through activating Wnt/beta-catenin signaling inhibitors DKK1 and SFRP2, Gynecol Oncol 147 (2017), 408–417.

21.

Fatima

et al., The natural compound Jatrophone interferes with Wnt/beta-catenin signaling and inhibits proliferation and EMT in human triple-negative breast cancer, Plos One 12 (2017), e189864.

22.

et al., Candidate tumor suppressor ZNF154 suppresses invasion and metastasis in NPC by inhibiting the EMT via Wnt/beta-catenin signalling, Oncotarget 8 (2017), 85749–85758.

23.

Ding

et al., MicroRNA-27a contributes to the malignant behavior of gastric cancer cells by directly targeting PH domain and leucine-rich repeat protein phosphatase 2, J Exp Clin Cancer Res 36 (2017), 45.

24.

Qiao

et al., MicroRNA-27a-3p modulates the Wnt/beta-catenin signaling pathway to promote epithelial-mesenchymal transition in oral squamous carcinoma stem cells by targeting SFRP1, Sci Rep 7 (2017), 44688.

25.

Tang

et al., miR-27a regulates endothelial differentiation of breast cancer stem like cells, Oncogene 33 (2014), 2629–2638.

26.

et al., MiRNA-27a promotes the proliferation and invasion of human gastric cancer MGC803 cells by targeting SFRP1 via Wnt/beta-catenin signaling pathway, Am J Cancer Res 7 (2017), 405–416.

27.

Zhou

et al., MiR-27a-3p functions as an oncogene in gastric cancer by targeting BTG2, Oncotarget 7 (2016), 51943–51954.

28.

Yeung

T.L.

et al., ELF3 is a negative regulator of epithelial-mesenchymal transition in ovarian cancer cells, Oncotarget 8 (2017), 16951–16963.

29.

Park

et al., FOXO1 Suppression is a determinant of acquired lapatinib-resistance in HER2-positive gastric cancer cells through MET upregulation, Cancer Res Treat 50 (2018), 239–254.

30.

Niimi

et al., Transcription factor FOXO1 promotes cell migration toward exogenous ATP via controlling P2Y1 receptor expression in lymphatic endothelial cells, Biochem Biophys Res Commun 489 (2017), 413–419.

31.

Stenman

et al., Suppression of forkhead box protein O1 (FOXO1) transcription factor may promote adrenocortical tumorigenesis, Horm Metab Res 49 (2017), 631–637.

32.

Yamamoto

et al., Lower FOXO3 mRNA expression in granulosa cells is involved in unexplained infertility, J Obstet Gynaecol Res 43 (2017), 1021–1028.

33.

et al., FoxO1 promotes mitophagy in the podocytes of diabetic male mice via the PINK1/parkin pathway, Endocrinology 158 (2017), 2155–2167.

34.

Deng

et al., Role of FoxO1 and apoptosis in pulmonary vascular remolding in a rat model of chronic thromboembolic pulmonary hypertension, Sci Rep 7 (2017), 2270.

35.

et al., Detection of FOXO1 break-apart status by fluorescence in situ hybridization in atypical alveolar rhabdomyosarcoma, Sci China Life Sci 60 (2017), 721–728.

36.

Wang

et al., Chitosan degradation products promote nerve regeneration by stimulating schwann cell proliferation via miR-27a/FOXO1 axis, Mol Neurobiol 53 (2016), 28–39.

37.

et al., High ALDH activity defines ovarian cancer stem-like cells with enhanced invasiveness and EMT progress which are responsible for tumor invasion, Biochem Biophys Res Commun 495 (2018), 1081–1088.

38.