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Introduction

Ovarian cancer is one of the most common solid tumors amongst women, with high mortality rates and few treatment options (1,2). Despite significant advances in ovarian cancer detection and research towards reducing recurrence rates, the five-year survival rate for patients with ovarian cancer has remained relatively stable for >20 years (1,2). The main obstacle for ovarian cancer therapy is disease recurrence, which is characterized by metastasis (3). For these reasons, the development of effective therapeutic strategies for ovarian cancer metastasis is urgently required.

The deregulation of oncogenes and tumor suppressors is closely associated with ovarian cancer development and progression (4). MicroRNAs (miRNAs), 18–25 nucleotide non-coding RNAs, bind to the 3′-untranslated region (UTR) of their target messenger RNAs (mRNAs), which results in the inhibition of translation or directly induces mRNA degradation (5). The deregulation of miRNA expression has been implicated in the development and progression of ovarian cancer (6). Of these deregulated miRNAs, miR-148a functions as a tumor suppressor in various types of cancer, including gastric, colorectal and non-small cell lung cancer (7–9). In addition, the expression of miR-148a was found to be reduced in ovarian cancer tissues and cell lines, and overexpression of miR-148a markedly inhibited cell proliferation in ovarian cancer cells, suggesting that the involvement of miR-148a in the carcinogenesis of ovarian cancer may occur via deregulation of cell proliferation (10). However, whether miR-148a has effects on ovarian cancer cell migration and/or invasion has remained to be elucidated, and the specific targets of miR-148a in ovarian cancer have not previously been studied.

Sphingosine-1-phosphate (S1P) has been found to have a critical role in the regulation of growth, metastasis and drug resistance in human malignancies, via binding to S1P receptors (S1PRs) and activating the downstream signaling pathways (11). S1PR1 is a member of the S1PRs, and S1P exerts pro-survival and drug resistant effects on cancer cells through binding to S1PR1 (12). However, the association between S1PR1 and miR-148a in ovarian cancer has remained to be determined.

In the present study, the expression levels of miR-148a in ovarian cancer tissues and cell lines, as well as in normal ovarian tissues and ovarian epithelial cells were evaluated. In addition, the effects of miR-148a on the migration and invasion of ovarian cancer cells, as well as the underlying molecular mechanisms were investigated.

Materials and methods

Reagents

Dulbecco's modified Eagle's medium (DMEM), TRIzol, fetal bovine serum (FBS), miRNA Reverse Transcription kit and Lipofectamine® 2000 were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). An miRNA Q-PCR Detection kit was purchased from GeneCopoeia, Inc. (Rockville, MD, USA). Mouse anti-S1PR1 monoclonal antibody (cat. no. ab72806), mouse anti-GAPDH monoclonal antibody (cat. no. ab8245) and rabbit anti-mouse immunoglobulin G secondary antibody (cat. no. ab175743) were purchased from Abcam (Cambridge, UK). An enhanced chemiluminescence (ECL) kit was purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA). A Quick-Change Site-Directed Mutagenesis kit was purchased from Stratagene (Agilent Technologies, Inc., Santa Clara, CA, USA). The psiCHECK™2 vector was purchased from Promega Corp. (Madison, WI, USA).

Tissue specimen collection

The study protocols were approved by the Ethics Committee of Xinxiang Medical University (Xinxiang, China). Informed consent was obtained from all patients with ovarian cancer recruited for the present study. Twenty ovarian cancer tissues, as well as their matched normal adjacent tissues, were collected at the Department of Gynecology and Obstetrics, The First Affiliated Hospital of Xinxiang Medical University (Weihui, China). Following surgical resection, the tissues were immediately snap-frozen in liquid nitrogen until use.

Cell culture

Human ovarian cancer cell lines, SKOV3, OVCAR and A2780, and normal ovarian epithelial cell line HUM-CELL-0088 were purchased from Nlunbio (Changsha, China). Cells were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified incubator containing 5% CO2.

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

Total RNA was extracted from the tissues and cells using TRIzol reagent, according to the manufacturer's instructions. An miRNA Reverse Transcription kit was used to convert RNA (1 µg) into cDNA, according to the manufacturer's instructions. Subsequently, PCR was performed using a miRNA Q-PCR Detection kit on an ABI 7500 Thermocycler (Applied Biosystems Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. The PCR conditions were as follows: 50°C for 2 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. The U6 gene was used as an internal reference. The relative expression was analyzed using the 2−ΔΔCt method.

Western blot analysis

The tissues or cells were solubilized in cold radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). Proteins were subsequently separated with 12% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane (Life Technologies, Carlsbad, CA, USA) and incubated with Tris-buffered saline with 5% Tween-20 (Sigma-Aldrich) containing 5% milk at room temperature for 3 h. The PVDF membrane was then incubated with mouse anti-S1PR1 antibody (1:100) and mouse anti-GAPDH antibody (1:50), respectively, at room temperature for 3 h. Following washing three times with phosphate-buffered saline Tween-20, the PVDF membrane was incubated with rabbit anti-mouse secondary antibody (1:5,000) at room temperature for 1 h. An ECL kit was used to perform chemiluminescent detection, and the relative protein expression levels were analyzed with Image-Pro Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). GAPDH was used as an internal reference.

Transfection

Transfection was performed using Lipofectamine 2000, according to the manufacturer's instructions. For miR-148a functional analysis, SKOV3 cells were transfected with scrambled miRNA as a negative control, miR-148a mimic or miR-148a inhibitor (all from Invitrogen Life Technologies), respectively. For S1PR1 functional analysis, SKOV3 cells were transfected with S1PR1-specific short interfering (si)RNA or S1PR1 plasmid (all from GenePharma, Co., Ltd, Shanghai, China), respectively.

Dual luciferase reporter assay

In accordance with the manufacturer's instructions, a Quick Change Site-Directed Mutagenesis kit was used to generate a mutant 3′-UTR of S1PR1. The wild-type or mutant 3′-UTR of S1PR1 was then inserted into the psiCHECK™2 vector by restriction enzyme digestion using XhoI and EcoRI (New England Biolabs, Ipswich, MA, USA). The plasmid was then ligated using T4 DNA ligase (New England Biolabs). SKOV3 cells were cultured to ~70% confluence, and were subsequently transfected with psiCHECK™2-S1PR1-3′-UTR or psiCHECK™2-mutant S1PR1-3′-UTR vector, with or without 100 nM miR-148a mimic, respectively. Following 48 h of transfection, luciferase activity was determined using an LD400 luminometer (Beckman Coulter, Brea, CA, USA). Renilla luciferase activity was normalized to firefly luciferase activity.

Migration assay

A Corning-Costar 3494 Transwell (Corning Life Sciences, Oneonta, NY, USA) was used to analyze SKOV3 cell migration. Briefly, a cell suspension (5×105 cells/ml) was prepared in serum-free DMEM. For each group, DMEM supplemented with 10% FBS was added to the lower chamber, and the cell suspension was added into the upper chamber. Following incubation for 24 h, cells that had not migrated through the membrane were removed. Cells that had migrated through the membrane were stained with 0.1% crystal violet, rinsed in water and air-dried. Six fields were randomly selected under the microscope (TS100; Nikon Corp., Tokyo, Japan), and the number of stained cells within these fields was counted.

Invasion assay

In order to evaluate cell invasion, 24-well Transwell chambers (Bioscience Research Reagents; Merck Millipore, Temecula, CA, USA) containing a layer of matrix gel were used. A cell suspension (5×105 cells/ml) was prepared in serum-free media. The cell suspension was added into the upper Transwell chamber, and DMEM with 10% FBS was added into the lower chamber. Following incubation for 24 h, the non-invading cells and the matrix gel on the interior of the inserts were removed. Cells on lower surface were stained with 0.1% crystal violet. Six microscopic fields were randomly selected and the number of stained cells was determined under a microscope (Nikon Corp.).

Statistical analysis

Values are expressed as the mean ± standard deviation of three independent experiments. Statistical analysis of differences was performed by one-way analysis of variance using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

miR-148a is downregulated in ovarian cancer tissues and cell lines

RT-qPCR was performed in order to determine the expression levels of miR-148a in twenty ovarian cancer tissue specimens and their matched normal adjacent tissues, three ovarian cancer cell lines and one normal ovarian epithelial cell line. As shown in Fig. 1A, miR-148a expression in ovarian cancer tissues was significantly downregulated, compared with that of their matched normal adjacent tissues. In addition, miR-148a expression was downregulated in the three ovarian cancer cell lines, SKOV3, OVCAR and A2780, compared with that of the HUM-CELL-0088 normal ovarian epithelial cell line (Fig. 1B). SKOV3 cells were chosen for subsequent experiments as they showed the most significant decrease in miR-148a expression levels among the three ovarian cancer cell lines.

Figure 1 miR-148a expression is downregulated in ovarian cancer tissues and cell lines. (A) RT-qPCR was performed to examine the relative expression of miR-148a in ovarian cancer tissues (Tumor) and their matched normal adjacent tissues (Normal). **P<0.01 vs. Normal. (B) RT-qPCR was performed to examine the relative expression of miR-148a in three ovarian cancer cell lines, SKOV3, OVCAR and A2780, and the normal ovarian epithelial cell line HUM-CELL-0088. **P<0.01 vs. HUM-CELL-0088. Values are expressed as the mean ± standard deviation. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR-148a, microRNA-148a.

S1PR1 expression is upregulated in ovarian cancer tissues and cell lines

A western blotting assay was performed to examine the protein expression of S1PR1 in the aforementioned tissues and cells. As shown in Fig. 2A, the protein levels of S1PR1 were markedly increased in ovarian cancer tissues, compared with those in their matched normal adjacent tissues. Concurrently, S1PR1 expression was also upregulated in ovarian cancer cells compared with that of normal ovarian epithelial cells (Fig. 2B).

Figure 2 S1PR1 expression is upregulated in ovarian cancer tissues and cells lines. (A) Western blotting was performed to determine the relative protein expression of S1PR1 in twenty ovarian cancer tissues (T) as well as their matched normal adjacent tissues (N). Three representative tumor tissues (T1, T2 and T3) and their matched normal adjacent tissues (N1, N2 and N3) are shown. **P<0.01 vs. normal tissues. (B) Western blot analysis was performed to determine the relative protein expression of S1PR1 in three ovarian cancer cell lines, SKOV3, OVCAR and A2780, and a normal ovarian epithelial cell line HUM-CELL-0088. **P<0.01 vs. HUM-CELL-0088. Values are expressed as the mean ± standard deviation. S1PR1, sphingosine-1-phosphate receptor 1.

miR-148a negatively regulates the protein expression of S1PR1 in SKOV3 cells

As miR-148a was downregulated while S1PR1 was upregulated in ovarian cancer cells and tissues, the association between these two factors was further examined in SKOV3 ovarian cancer cells. Following transfection of SKOV3 cells with scrambled miRNA, miR-148a mimic or miR-148a inhibitor, respectively, the expression levels of miR-148a in each group were determined, and the results indicated that the transfection efficiency was satisfactory (Fig. 3A). The protein expression levels of S1PR1 in each group were also determined, and the results indicated that S1PR1 was significantly downregulated following overexpression of miR-148a, but was upregulated following inhibition of miR-148a in SKOV3 cells (Fig. 3B), suggesting that miR-148a may negatively regulate the protein expression of S1PR1 in SKOV3 ovarian cancer cells.

Figure 3 miR-148a negatively regulates S1PR1 protein expression in ovarian cancer cells. (A) Reverse transcription-quantitative polymerase chain reaction was performed to determine the relative expression of miR-148a in SKOV3 cells transfected with scrambled miRNA, miR-148a mimics and miR-148a inhibitor. Control, SKOV3 cells without any transfection. (B) Western blot analysis was performed to examine the protein levels of S1PR1 in SKOV3 cells transfected with scrambled miRNA, miR-148a mimics and miR-148a inhibitor. Control, SKOV3 cells without any transfection. **P<0.01 vs. Control. Values are expressed as the mean ± standard deviation. miRNA, microRNA; miR-148a, microRNA-148a; S1PR1, sphingosine-1-phosphate receptor 1; NC, scrambled miRNA.

miR-148a inhibits SKOV3 cell migration and invasion via targeting S1PR1

The roles of miR-148a and S1PR1 in the regulation of cell migration and invasion in SKOV3 cells were further evaluated. The results revealed that overexpression of miR-148a or inhibition of S1PR1 suppressed SKOV3 cell migration and invasion (Fig. 4). However, overexpression of S1PR1 reversed the inhibitory effect of miR-148a overexpression on SKOV3 cell migration and invasion. These data suggested that miR-148a suppressed SKOV3 cell migration and invasion, at least in part, via inhibition of S1PR1.

Figure 4 miR-148a inhibits ovarian cancer cell migration and invasion. (A) A cell migration assay was performed to determine the migration capacity of SKOV3 cells transfected with miR-148a mimics, S1PR1 siRNA or co-transfected with miR-148a mimics and S1PR1 plasmid (0.1% crystal violet, ×200 magnification). (B) A cell invasion assay was performed to determine the cellular invasion capacity of SKOV3 cells transfected with miR-148a mimics, S1PR1 siRNA or co-transfected with miR-148a mimics and S1PR1 plasmid (0.1% crystal violet, ×200 magnification). **P<0.01 vs. Control. Values are expressed as the mean ± standard deviation. Control, SKOV3 cells without any transfection. miR-148a, microRNA-148a; S1PR1, sphingosine-1-phosphate receptor 1; siRNA, short interfering RNA.

S1PR1 is a novel target of miR-148a in SKOV3 cells

In order to investigate whether S1PR1 was a target of miR-148a, wild-type and mutant S1PR1 3′-UTR were generated (Fig. 5A). Subsequently, a luciferase reporter assay was performed in SKOV3 cells, and luciferase activity was demonstrated to be significantly downregulated in SKOV3 cells co-transfected with the wild-type 3′UTR of S1PR1 and miR-148a mimic (Fig. 5B). However, luciferase activity remained unchanged in the SKOV3 cells co-transfected with mutant S1PR1 3′UTR and miR-148a mimic. These data indicated that S1PR1 may be a direct target of miR-148a in SKOV3 cells.

Figure 5 S1PR1 is a novel target of miR-148a in SKOV3 cells. (A) Seed sequences of miR-148a in the WT and MUT 3′-UTR of S1PR1. (B) Luciferase reporter assay data revealed that luciferase activity was downregulated in SKOV3 cells co-transfected with miR-148a and WT S1PR1 3′-UTR, and unaltered in SKOV3 cells co-transfected with miR-148a and MUT S1PR1 3′-UTR. Luciferase activity was also unaltered in SKOV3 cells co-transfected with NC miRNA and WT or MUT S1PR1 3′-UTR. Control, SKOV3 cells co-transfected with blank vector and WT or MUT S1PR1 3′-UTR. **P<0.01 vs. Control. Values are expressed as the mean ± standard deviation. miR-148a, microRNA-148a; WT, wild-type; MUT, mutant; 3′-UTR, 3′-untranslated region; S1PR1, sphingosine-1-phosphate receptor 1; NC, negative control; miRNA, microRNA.

Discussion

The results of the present study indicated that miR-148a expression was markedly downregulated in ovarian cancer tissues and cell lines, compared with that of the normal adjacent tissues and ovarian epithelial cells, respectively. S1PR1 was further identified as a direct target of miR-148a, and the protein expression of S1PR1 was significantly upregulated in ovarian cancer tissues and cell lines. In addition, it was demonstrated that S1PR1 expression was negatively regulated by miR-148a. Investigation into the underlying molecular mechanisms revealed that the inhibitory effect of miR-148a on ovarian cancer cell migration and invasion was via direct targeting of S1PR1.

miRNAs are able to negatively regulate gene expression via inhibition of mRNA translation or induction of mRNA degradation. The deregulation of miRNA expression is a key molecular mechanism by which the expression levels of oncogenes or tumor suppressors are mediated in cancer cells (13). Zhou et al (10) examined the expression levels of miR-148a in 78 patients with epithelial ovarian cancer, 17 normal ovarian epithelium tissues and two ovarian cancer cell lines. The results revealed that the expression of miR-148a was markedly reduced in ovarian cancer tissues and cell lines (10), consistent with the results of the present study. Furthermore, miR-148a mimics were transfected into ovarian cancer cell lines, and the results demonstrated that the upregulation of miR-148a inhibited ovarian cancer cell proliferation (10). Accordingly, miR-148a may represent a novel biomarker for early detection or therapeutic targets of ovarian cancer. However, to the best of our knowledge, the specific role of miR-148a in the regulation of cell migration and invasion in ovarian cancer cells, as well as the underlying molecular mechanisms, has not previously been reported.

The effects of miR-148a on cell migration and invasion in other types of cancer have been demonstrated. For example, Zheng et al (14) revealed that the overexpression of miR-148a inhibited gastric cancer cell migration and invasion in vitro and lung metastasis formation in vivo. Another study reported that miR-148a overexpression inhibited cell migration and invasion in prostate cancer cells (15). In the present study, it was demonstrated that restoration of miR-148a expression significantly suppressed cell migration and invasion in ovarian cancer SKOV3 cells. Based on the above findings, it was hypothesized that the inhibitory effects of miR-148a on cancer cell migration and invasion may be universal.

The underlying molecular mechanisms by which miR-148a mediated ovarian cancer cell migration and invasion were further investigated, and the results indicated that S1PR1 was involved in the miR-148a-mediated inhibition of cell migration and invasion in ovarian cancer SKOV3 cells. S1P has been found to be aberrantly expressed in patients with ovarian cancer, and is involved in the regulation of key cellular processes that contribute to the development and progression of ovarian cancer (16,17). In addition, agents that block the S1P/S1PRs signaling pathway were reported to inhibit ovarian cancer cell growth or induce apoptosis (18). Furthermore, the S1P/S1PRs signaling pathway has been found to be involved in the regulation of ovarian cancer invasion potential (19). Wang et al (17) revealed that physiological concentrations of S1P promoted the migration and invasion of ovarian cancer cells but inhibited the migration of human ovarian epithelial cells. In addition to the effects of S1P in SKOV3 cells, S1P was also found to induce the invasion of OVCAR3 ovarian cancer cells, an effect which was inhibited by VPC 23019, an antagonist of S1PR1 (20). The results of the present study indicated that the expression of S1PR1 was markedly increased in ovarian cancer tissues and cell lines, compared with that of normal ovarian tissues and epithelial cells. Another study indicated that S1PR1 was expressed in the hen and human ovary, as well as in ovarian tumors (21). Furthermore, S1PR1 was identified as a novel target of miR-148a, and its expression was negatively regulated by miR-148a in ovarian cancer cells. These findings further confirmed the association between miR-148a and S1PR1 in ovarian cancer cells.

In conclusion, the results of the present study revealed that miR-148a had an inhibitory effect on migration and invasion in ovarian cancer cells, at least in part, via direct inhibition of S1PR1, a novel target of miR-148a. Therefore, miR-148a may potentially be used as a molecular agent for the prevention and treatment of invasion and metastasis in ovarian cancer, while S1PR1 may present a promising target for clinical applications.

References