The present study aimed to investigate the potential role of microRNA (miR)-214 in targeting the phosphatase and tensin homolog (PTEN)-mediated phosphoinositide 3-kinase (PI3K)/Akt signaling pathway in ovarian cancer (OC). The target gene of miR-214 was determined by luciferase reporter gene assay and was indicated to be PTEN. Human SK-OV-3 cells were transfected with a miR-214 inhibitor and a miR-214 mimic, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect relative expression of miR-214. The MTT assay was performed to detect cell viability following transfection. Cell cycle and apoptosis were assessed by staining with propidium iodide (PI) and double staining with Annexin V/PI, respectively. The expression levels of PTEN and PI3K/Akt signaling pathway-associated proteins were detected by western blot analysis. The expression of miR-214 in tumor tissues and normal tissues was detected by RT-qPCR, and PTEN expression was detected by immunohistochemistry. SK-OV-3 cells transfected with a miR-214 inhibitor showed significantly inhibited cell viability and proliferation, and markedly increased apoptotic rate. SK-OV-3 cells transfected with miR-214 mimic showed significantly increased viability and proliferation, and markedly decreased apoptotic rate. The cells transfected with a miR-214 inhibitor exhibited significantly upregulated PTEN expression and significantly downregulated phosphatidylinositol (
As the fifth leading cause of cancer-associated mortalities resulting from gynecological malignancies, ovarian cancer (OC) presented with the highest incidence rates in Europe and North America, and lowest in Africa and Asia in 2008. Additionally, an estimated 22,240 new cases and 14,030 mortalities were reported in the United States and ~4,500 mortalities per year in the UK (
Accumulating evidence revealed significant roles of miRs in various human cancers by modulating several biological and pathological processes, including differentiation, growth, proliferation and apoptosis in cells, as well as angiogenesis, invasion and metastasis in tumors (
The SK-OV-3 (human ovarian cancer) cell line was provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). SK-OV-3 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), and incubated in a 5% CO2 incubator at 37°C. Cell subcultures were performed when cells were grown to 90% confluence. The primary medium was subsequently discarded. The cells were washed twice with phosphate-buffered saline (PBS), digested with trypsin (Gibco; Thermo Fisher Scientific, Inc.) and treated with the RPMI-1640 medium containing 10% FBS to form a single-cell suspension. Routine subculture was performed. The cells in logarithmic phase were collected for subsequent usage.
DNA extraction was performed with the TIANamp Genomic DNA kit (Tiangen Biotech Co., Beijing, China) according to the manufacturer's protocol. In order to investigate whether binding sites predicted by miR-214 induced changes in luciferase activity, PTEN-mutation (PTEN-mut; 5′-TTA-TTT TAC TAG TTT TCA ATC ATA ATA CCT GAC AT-3′) and wide-type PTEN (PTEN-wt; 5′-TTA-TTT TAC TAG TTT TCA ATC ATA ATA CCT GCT GT-3′) both from Synbio Technologies (Jiangshu, China), were designed and inserted into a reporter plasmid, which was purchased from Miaolingbio, Inc. (Hubei, China). PTEN and the corresponding miR-214 binding target site were determined using a target prediction software (Target Scan) (
The cells were divided into four groups: Blank (cells untransfected with any miR-214 sequence), negative control (NC), miR-214 mimic and miR-214 inhibitor. The cells in the NC group were transfected with a vector containing the miR-214 NC sequence (5′CCU GAC AAU UAG UAU UU-3′; Shanghai GenePharma Co., Ltd., Shanghai, China) and the cells in the miR-214 mimic group were transfected with a miR-214 mimic (5′-ACAGGUAGCUGAACACUGGGUU-3′; Synbio Technologies). The cells in the miR-214 inhibitor group were transfected with mirVana™ miRNA inhibitor (5′-UCACAGUGCUCAUCAUGAAUAA-3′; Shanghai Bioleaf, Shanghai, China). SK-OV-3 cells (200 µl/well) in the logarithmic growth phase were seeded on a 6-well plate with antibiotic-free complete medium. SK-OV-3 cells were transfected with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) when cell density reached 30–50%, according to the manufacturer's instructions. The miR-214 NC vector, miR-214 mimic and a miR-214 inhibitor (100 pmol; final concentration, 50 nM) were respectively diluted in 250 µl serum-free medium Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.), gently mixed until even and incubated for 5 min at room temperature. Lipofectamine 2000 (5 µl) was diluted in 250 µl serum-free medium Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.), gently mixed until even, and incubated for 5 min at room temperature. The diluted miR-214 NC vector, miR-214 mimic and miR-214 inhibitor were evenly mixed with the diluted Lipofectamine 2000, respectively. The mixture was added into the well containing cells following incubation for 20 min at room temperature and gently mixed until even. The transfected cells were placed in a 5% CO2 incubator at 37°C. The medium was replaced with complete medium after 6–8 h incubation at 37°C, and the transfected cells were incubated at 37°C for 24–48 h for subsequent experiments.
The cells (80% confluence) were washed twice with PBS and digested with 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.) to form a single-cell suspension. The cells were counted using Moxi Z mini automated cell counter (Bio Excellence International Tech Co., Ltd., Beijing, China), according to the manufacturer's instructions and were inoculated in a 96-well plate (200 µl per well, 6 repeated wells) at a density of 3×103 to 6×103. Following culture at 37°C for 48 h, 5 mg/ml MTT solution (20 µl) was added to each well. After 4 h incubation at 37°C, the culture medium was discarded. Dimethyl sulfoxide (150 µl; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to each well and gently agitated for 10 min. Optical density (OD) values were determined at a wavelength of 490 nm with an enzyme-linked immunosorbent detector (De Tie Inc., Nanjing, China) at 12, 24 and 48 h, respectively. Cell viability curves were plotted with time as the x-axis and the OD value as the y-axis.
Following a 48-h transfection at 37°C, the cells were digested with the EDTA-free trypsin and collected in the flow tube for centrifugation at 157 × g at 4°C for 5 min, and the supernatant was discarded. Cells were washed three times with PBS, and subsequently centrifuged at 157 × g at 4°C for 5 min for removal of supernatant. According to the manufacturer's instructions, 150 µl binding buffer and 5 µl (per tube) Annexin V-FITC [Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit; Sigma-Aldrich; Merck KGaA] was added to the cells and gently agitated. The cells were incubated in darkness for 15 min at room temperature, and an additional 100 µl binding buffer and 5 µl propidium iodide (PI) dye (Sigma-Aldrich; Merck KGaA) was added. The mixture was oscillated and mixed evenly. The proportion of cells undergoing apoptosis was analyzed by CytoFLEX flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) and the results were analyzed using CytExpert 2.0 software (Beckman Coulter, Inc.).
Following a 48-h transfection at 37°C, the cells were collected, washed with cold PBS for three times and centrifuged at 157 × g at 4°C for 5 min to remove the supernatant. Cell concentration was adjusted to ~1×105/ml, and the cells were fixed with 1 ml 75% ice-cold ethanol overnight at 4°C. The cells were subsequently washed twice with PBS with supernatant discarded prior to staining and 100 µl RNase A was then added in darkness. The cells were incubated in a 37°C water bath for 30 min, stained with 400 µl PI and mixed evenly. Subsequently, the samples were incubated in the dark at 4°C for 30 min. Cell cycle was analyzed by detecting red fluorescence at excitation wavelength of 488 nm with the CytoFLEX flow cytometer (Beckman Coulter, Inc.).
After 48 h of transfection, the cells were collected, lysed by Tissue Total Protein Lysis Buffer Solution (Shanghai Yu Bo Biological Technology Co., Ltd., Shanghai, China) and centrifuged. The bicinchoninic acid kit (Thermo Fisher Scientific, Inc.) was used following the manufacturer's instructions to measure total protein concentration. The total protein (20 µg) was run on a 12% SDS-PAGE and electro-transferred to polyvinylidene fluoride membranes. The membranes were blocked with skimmed milk at room temperature for 1 h, incubated with monoclonal antibodies GAPDH (GTX101025, 1:500), PTEN (GTX101025, 1:500), PIP3 (GTX119163, 1:500), Akt (GTX128414, 1:500), phosphorylated (p)-Akt (GTX121937, 1:500), glycogen synthase kinase (GSK)-3β (GTX111192, 1:500) and p-GSK-3β (GTX132997, 1:500; all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. The membrane was washed with PBS three times to remove primary antibodies and incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (GTX77266, 1:500; Beyotime Institute of Biotechnology, Haimen, China) at room temperature for 1–2 h, washed with PBS three times, and an electro-chemiluminescence (ECL) color developing kit (Shanghai Yeasen Biotechnology Co., Ltd., Shanghai, China) for visualization of the bands, according the manufacturer's instructions. Images were captured using an
The specimens were obtained from 124 patients with OC who underwent surgical treatment in Linyi Tumor Hospital (Linyi, China) between February 2013 and February 2015. All the specimens were confirmed by pathological diagnosis. All patients were females with childbearing history (the mean age, 48.17±10.32 years; range, 34–73 years), who had received no preoperative chemotherapy. Surgical-pathological staging (
Analysis of miR-214 expression in OC tissues and adjacent normal tissues was performed with RT-qPCR. The total RNA was extracted from frozen tissues (50 mg) with a miRNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA) following the manufacturer's instructions. RNA sample (5 µl) was diluted (20X) with RNase-free ultra-pure water (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The adsorptions of UV light at wavelengths 260 and 280 nm (OD260/OD280 ratio) were read from the ultraviolet spectrophotometer (Mettler Toledo Instruments, Shanghai, China) to determine purity and contamination of the RNA samples. Only high-purity RNA samples with an OD260/OD280 ratio of 1.7–2.1 were used for subsequent analysis. Complementary DNA (cDNA) was obtained by reverse transcription with a PCR amplification instrument. A total of 9.0 µl diethyl pyrocarbonate (DEPC)-treated water, 0.6 µl reverse primers (20 pmol/µl), 0.1 µl enzyme inhibitor (40 U/µl) and 5 µl RNA solution were added to a microcentrifuge tube (0.5 ml, RNase-free). Following incubation in a water bath at 70°C for 5 min and in an ice bath for 5 min, the mixture was incubated with 4.0 µl 5xAMV reverse transcription buffer, 1.0 µl dNTP Mixture (10 mol/l), 0.1 µl RNA enzyme inhibitor (40 U/µl) and 0.2 µl AMV Reverse Transcriptase (5 U/µl) at 42°C for 40 min, followed by amplification using a PCR instrument. RT-qPCR experiment was performed using a ABI7500 quantitative PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following PCR conditions: Pre-denaturing (95°C, 10 min); followed by 40 cycles of denaturing at 95°C for 10 sec and annealing at 60°C for 20 sec; and extension at 72°C for 34 sec. Real-time detection was performed using SYBR-Green (Thermo Fisher Scientific, Inc.) with maximum absorption wavelength 497 nm and emission maximum wavelength 520 nm for fluorescence signals. The sequences of the primers used are as follows: miR-214 forward, 5′-CACCTTTCTCCCTTTCCCCTTACTCTCC-3′ and reverse, 5′-TTTCATAGGCACCACTCACTTTAC-3′; U6 (internal control) forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The 2−ΔΔCq method (
The 10% formalin fixed and paraffin-embedded specimens (n=62) were cut into 4 µm-thick sections. The tissue slices were dried in an oven at 60°C for 1 h, dewaxed with xylene, dehydrated in graded ethanol (50, 70, 80 and 95%) and incubated with 3% H2O2 (Sigma-Aldrich; Merck KGaA) at 37°C for 30 min, washed with PBS and placed in 0.01 M citrate buffer, boiled at 95°C for 20 min, cooled to room temperature and washed with PBS. The washed tissue sections were incubated at 37°C for 10 min in blocking medium containing normal goat serum (Wak-Chemie Medical GmbH, Steinbach, Germany), incubated with the primary antibody (cat. no. GTX101025; PTEN; 1:100; Santa Cruz Biotechnology, Inc.) overnight at 4°C, rinsed with PBS, and additionally incubated with the horseradish peroxidase-labeled secondary antibody (GTX77266, 1:500, Beijing Biosynthesis Biotechnology Co., Ltd.) for 30 min at room temperature. The sections were visualized using 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich; Merck KGaA) followed by staining with hematoxylin (5 mg/ml; Shanghai Bogoo Biotechnology. Co., Ltd., Shanghai, China) and mounting. A negative control was prepared by replacing the primary antibody with PBS, and normal mucosa tissues were used as a positive control. The expression of PTEN was mainly observed in the cell nucleus and partially in the cytoplasm, presenting with yellow or brownish-yellow granules. A total of five high-power fields (×400, 100 counted cells per field) using a CX31-LV320 OLYMPUS microscope (Chang Heng Rong technology Co., Ltd., Beijing, China) in each tissue section were randomly selected, and positive cells/all tumor cells >10% was considered as positive (+) and positive cells ≤10% as negative (−). The results of immunohistochemistry were evaluated independently by two experienced observers using a double-blind approach.
Continuous variables are presented as the mean ± standard deviation. The comparison between the two groups was performed using paired t-test, and the comparison among the groups was analyzed using one-way analysis of variance. Categorical variables are presented as frequencies and percentages, and comparisons were performed with χ2 test. SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. P<0.05 was considered to indicate a statistically significant difference.
The sequences of miR-214 binding with 3′-UTR region of PTEN mRNA were illustrated in
RT-qPCR results indicated that the expression of miR-214 decreased significantly in the SK-OV-3 cells transfected with a miR-214 inhibitor compared with the untransfected cells (blank) and NC groups. By contrast, there was a significant increase in miR-214 expression in the miR-214 mimic group compared with the blank (all P<0.05). miR-214 expression was markedly lower in the miR-214 inhibitor group compared with the miR-214 mimic group (P<0.05). There were no significant differences in miR-214 expression between the blank and NC P>0.05;
MTT assay indicated that, cell viability was significantly inhibited in the miR-214 inhibitor group compared with the blank and the NC group (P<0.05). There were significant differences in OD values between the miR-214 inhibitor group and the blank group at the time points 24 and 48 h (P<0.05). There were also significant differences between the miR-214 inhibitor group and NC group (P<0.05). Cell viability increased significantly in the miR-214 mimic group compared with the blank and the NC group. Significant differences in OD values were observed between the miR-214 mimic group and the blank and the NC group at the time points 24 and 48 h (both P<0.05). Significantly increased OD values were observed in the miR-214 mimic group compared with the miR-214 inhibitor group at the time points of 24 and 48 h (P<0.05). The comparison of OD values between the blank and the NC group showed no significant difference at any time points (all P>0.05). With the increase in time, there were significant differences in OD among different groups [multivariate analysis of variance (F)=24.544; P<0.001]. There were significant differences in OD values between the different time points (F=217.932; P<0.001). Additionally, there was interaction between the measurement time and group (F=23.839; P<0.001;
PI staining results showed that the proportion of cells in G1 in the blank, NC, miR-214 mimic and miR-214 inhibitor groups were 56.39±3.82, 57.62±3.34, 60.34±2.38 and 76.40±2.22%, respectively (
Double staining with Annexin V and PI results indicated that 48 h following transfection, apoptotic rates in the blank, the NC, the miR-214 mimic group and the miR-214 inhibitor group were 11.4, 10.9, 4.9 and 28.1%, respectively. The apoptotic rate of SK-OV-3 cells in the miR-214 inhibitor group was significantly increased compared with the other three groups (all P<0.05). However, the apoptotic rate was not statistically significant between the blank group and the NC group (P>0.05;
Western blot analysis results revealed significantly increased expression of PTEN and decreased expression of PIP3, p-Akt and p-GSK-3β in SK-OV-3 cells transfected with a miR-214 inhibitor (48 h later) compared with the blank group and the NC group (all P<0.05). SK-OV-3 cells transfected with the miR-214 mimic exhibited significantly downregulated PTEN expression and significantly upregulated PIP3, p-Akt and p-GSK-3β expression compared with the blank and the NC group (all P<0.05). Significant differences in PTEN, PIP3, p-Akt and p-GSK-3β expression levels were observed between the miR-214 inhibitor group and the miR-214 mimic group (all P<0.05). There were no significant differences in Akt and GSK-3β among all groups (all P>0.05). The expression of each protein in the blank group did not significantly differ from those in the NC group (all P>0.05;
The results from RT-qPCR showed that OC tissues had a significantly increased expression of miR-214 in comparison with adjacent normal tissues (P<0.01;
The functional role of miR-214 in regulating PTEN-mediated PI3K/Akt signaling pathway was evaluated. It has been predicted that miR-214, which is located inside the sequence of long noncoding Dmn3os transcript, targets two activating protein 2 transcription factors, causing downstream effects on genes regulating vital cell cycle processes, including apoptosis, proliferation and angiogenesis (
Extensive roles of miR-214 in promoting tumor cell proliferation, growth and invasion, as well as tumor progression and metastasis have been previously reported (
In conclusion, OC tissues exhibited increased expression of miR-214 and decreased expression of PTEN protein compared with normal tissues. miR-214 may activate the PI3K/Akt signaling pathway by downregulating the targeted PTEN, which may promote proliferation and inhibit apoptosis of OC cells and provide a potential miRNA-based targeted therapy for the treatment of OC.
Predicted binding site of miR-214 in the PTEN. (A) Binding of miR-214 to the 3′-UTR of PTEN mRNA; (B) Detection of reporter gene activity based on the dual luciferase reporter system. SK-OV-3 cells were co-transfected with miR-214 mimic, Wt-miR-214/PTEN and Mut-miR-214/PTEN; miR-214 was able to significantly inhibit the luciferase activity of Wt-miR-214/PTEN plasmid but had no significant effect on the luciferase activity of Mut-miR-214/PTEN plasmid; *P<0.05 compared with the NC group. PTEN, phosphatase and tensin homolog; miR, microRNA; Mut, mutated; UTR, untranslated region; Wt, wild-type; NC, negative control.
miR-214 expression in SK-OV-3 cells transfected with a miR-214 inhibitor and a miR-214 mimic after 48 h of transfection; *P<0.05 compared with the blank and NC groups; #P<0.05 compared with the miR-214 mimic group; NC, negative control; miR, microRNA; blank, cells untransfected with miR-214.
Viability curves of SK-OV-3 cells transfected with miR-214 mimic and treated with miR-214 inhibitor following 48 h of incubation by MTT assay. miR-214 mimic significantly promoted the viability of SK-OV-3 cells, and miR-214 inhibitor significantly inhibited the viability of SK-OV-3 cells. NC, negative control; miR, microRNA; OD, optical density; blank, cells untransfected with miR-214.
Cell cycle analysis of SK-OV-3 cells transfected with miR-214 mimic and treated with a miR-214 inhibitor by propidium iodide staining. After 48 h of transfection, the majority of cells treated with a miR-214 inhibitor were in G1. By contrast, the proportion of miR-214 inhibitor-treated cells in the S phase was significantly decreased, and the proliferation of SK-OV-3 cells was significantly inhibited compared with cells in the blank, the NC and the miR-214 mimic group. *P<0.05 compared with the blank, NC and miR-214 mimic groups; NC, negative control; miR, microRNA; blank, cells untransfected with miR-214.
Analysis of apoptosis by double staining with Annexin V and PI. (A) Blank, (B) NC, (C) miR-214 mimic and (D) miR-214 inhibitor group. The cells were analyzed 48 h following transfection. NC, negative control; PI, propidium iodide; FITC, fluorescein isothiocyanate; miR-214, microRNA-214; blank, cells untransfected with miR-214.
Western blotting of PTEN and PI3K/Akt pathway-associated proteins. (A) Expression levels were analyzed by western blotting following 48-h of transfection. (B) PTEN, PIP3, (C) p-Akt, GSK-3β, (D) Akt and p-GSK-3β expression levels. There was a significant increase in expression of PTEN and a significant decrease in expression of PIP3, p-Akt and p-GSK-3β in SK-OV-3 cells transfected with a miR-214 inhibitor compared with the blank, NC and miR-214 inhibitors groups. There was a significantly downregulated PTEN expression and significantly upregulated PIP3, p-Akt and p-GSK-3β expression in miR-214 mimic-transfected cells compared with the blank, NC and miR-214 mimics groups. There were significant differences in PTEN, PIP3, p-Akt and p-GSK-3β expression between the miR-214 inhibitor group and the miR-214 mimic group. *P<0.05 compared with the blank and NC groups; #P<0.05 compared with miR-214 mimic group; sphatidylinositol (
Expression of miR-214 in ovarian cancer tissues and adjacent normal tissues. miR-214 expression was significantly increased in the ovarian cancer tissues compared with normal tissues. *P<0.05, compared with adjacent normal tissues; miR-214, microRNA-214; blank, cells untransfected with miR-214.
Expression of PTEN in ovarian cancer tissues and adjacent normal tissues PTEN. (A) Strong positive expression of PTEN in normal ovarian tissues. (B) Weak positive expression of PTEN in ovarian cancer tissues. Magnification, ×200; PTEN, phosphatase and tensin homolog.
Differences in the positive expression rate of PTEN protein in ovarian cancer and adjacent normal tissues.
PTEN expression, n | |||||
---|---|---|---|---|---|
Type of tissue | Number of cases | Positive | Negative | PTEN positive rate, % | P-value |
Ovarian cancer | 124 | 59 | 65 | 47.6 | <0.001 |
Normal tissues | 124 | 96 | 28 | 77.4 |
PTEN, phosphatase and tensin homolog deleted on chromosome 10; P<0.001, compared with the ovarian cancer tissues and the adjacent tissues.