A total of 30 patients with ovarian cancer who received their treatment in The Department of Obstetrics and Gynecology in The First Affiliated Hospital of Fujian Medical University from February 2013 to December 2014 were collected. None of the patients underwent chemotherapy and radiotherapy prior to surgery. The patients were aged from 33–72 years, with a mean age of 51.3±21.4 years. The tumor specimens and normal para-carcinoma tissue specimens were collected by surgery. Tumor tissue specimens were pathologically confirmed as ovarian cancer, whilst the normal tissues were confirmed as having no cancerous tissue invasion by two pathologists from the First Affiliated Hospital of Fujian Medical University. Specimens were preserved in liquid nitrogen within 10 min following acquisition. All specimens were collected with the patient's informed consent and approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University.

Ovarian cancer cell line SKOV3 cells were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences in Beijing, China. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (catalog no. SA00001) was purchased from OriGene Technologies, Inc. (Beijing, China). Annexin V/PI staining kits and caspase-3 enzyme activity test kits were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). TRIzol^® and transfection reagent Lipofectamine^® 2000 were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Mimic NC, miR-221 mimic, anti-miR-NC and anti-miR-221 were purchased from Guangzhou Ribobio Co., Ltd. (Guangzhou, China). Luciferase activity detection kits were purchased from Promega Corporation (Madison, WI, USA). Mouse anti-human β-actin (catalog no. MS123A1) and BMF antibodies (catalog no. A21422), and si-NC (catalog no. A24185) and si-BMF (catalog no. A36521) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The cell proliferation detection kit (Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit) was purchased from Molecular Probes (Thermo Fisher Scientific, Inc.). Overexpression plasmid phosphorylated internal ribosome entry site 2 (pIRES2) was purchased from Beijing BioVector (Beijing, China; http://www.biovector.net/).

A total of 1 ml TRIzol was added to every 20 mg of tissue or every 3×10⁶ cells. Following full lysis, chloroform was added for extraction. Following separation, the RNA supernatant was transferred to a new Eppendorf (EP) Tube^®, precipitated with isopropanol and washed with 70% ethanol, and then dissolved in diethyl pyrocarbonate water to extract RNA. The RNA was reverse transcribed into cDNA using the PrimeScript^™ RT reagent Kit (Perfect Real Time, Takara, Japan; URL: http://www.takara-bio.com/), then using cDNA as a template, qPCR was performed for detection of gene expression. The reverse transcription reaction system included 0.5 µl oligdT Primer (50 µM), 0.5 µl Random 6-mers (100 µM), 0.5 µl PrimeScript RT Enzyme Mix, 1.0 µg RNA, 2 µl 5X PrimeScript Buffer, then RNase-free water was added to a total volume of 10.0 µl. The reverse transcription reaction condition: 37°C for 15 min; and 85°C for 5 sec. The RT-qPCR reaction system included SYBR Fast qPCR Mix 10.0 µl, 0.8 µl forward Primer (10 µM), 0.8 µl Reverse Primer (10 µM), 2.0 µl cDNA and 6.4 µl RNase Free dH₂O. The qPCR reaction conditions: Initial denaturation 10 min at 95°C; denaturation 10 sec at 95°C; annealing 20 sec at 60°C; and extension 15 sec at 72°C; 40 cycles, and finally the gene expression was detected in ABI ViiA 7 real-time qPCR instrument. The 2^−∆∆Cq method was used to compare gene expression between clinical samples (23).

The primer sequence used were: miR-221P forward, 5′-GTTGGTGGGAGCTACATTGTCTGC-3′, and reverse, 5′-GTGTCGTGGACTCGGCAATTC-3′; U6P forward, 5′-ATTGGAACGATACAGAGAAGATT-3′, and reverse, 5′-GGAACGCTTCACGAATTTG-3′; BMFP forward, 5′-TTTATGGCAATGCTGGCTATCG-3′, and reverse, 5′-GCAATCTGTACCTCTGCTTGATG-3′; and β-actin forward, 5′-GAACCCTAAGGCCAAC-3′, and reverse, 5′-TGTCACGCACGATTTCC-3′.

The fragments of the coding region of the BMF gene were amplified and the size of target fragments was identified by gel electrophoresis (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). SDS PAGE gel (9%) was prepared as follows: 50 g of protein sample was loaded for 3 h and then placed into the electric transducers, in which the buffer was added to the transmembrane for 1.5 h and the target protein was transferred onto nitrocellulose membranes. PCR product was recovered from gel, and following dual-enzyme digestion they were ligated into pIRES2 plasmids, then following transformation the positive strains were selected, and strains were amplified and cultured to extract the recombinant plasmid containing the target fragments. Sequencing was performed to confirm that the target fragment of BMF gene was inserted correctly, and termed as pIRES2-BMF; in addition, the blank vector pIRES2-blank was used as control.

SKOV3 cells were cultured in DMEM containing 10% FBS and 1% streptomycin, and cultured in a cell incubator at 37°C and an atmosphere containing 5% CO₂. Medium was replaced once every two days. Cells were subcultured according to the ratio of 1:4; cells in good growth condition at the logarithmic period were taken to carry out experiments. One day prior to transfection, cells were inoculated in a 10 cm culture dish, to ensure that the cell density could be ≤50–60% on the day of transfection. A total of 2 µl Lipofectamine 2000, 30 nmol anti-miR-221p, 20 nmol pIRES2-BMF, and 20 nmol si-BMF were diluted into 50 ml Opti-MEM (Invitrogen; Thermo Fisher Scientific, Inc.) and added to the cells. Following incubation at room temperature for 5 min, the diluted Lipofectamine 2000 and all transfectants were gently mixed well and placed at room temperature for 20 min, then according to difference of the grouping, the transfected mixture was added to cells free of serum and double antibody culture medium (Invitrogen; Thermo Fisher Scientific, Inc.), incubated in 37°C incubator for 6 h, and then the medium was changed to fresh complete DMEM medium containing serum (Invitrogen; Thermo Fisher Scientific, Inc.) and double antibody, to continue to culture 48 h, and cells were collected for detection. The transfected cells were divided into four groups: Anti-miR-NC, anti-miR-221, pIRES2-blank and pIRES2-BMF.

The SKOV3 cells transfected with anti-miR-NC, anti-miR-221, pIRES2-blank and pIRES2-BMF were inoculated into 96-well culture plates. Following incubation for 24, 48 and 72 h at 37°C following transfection, 10 µl MTT solution (Qiagen GmbH, Hilden, Germany) was added to each well for 4 h at 37°C, the supernatant was removed, and 150 µl dimethyl sulfoxide solution was added, following vibration for 10 min at low speed, the absorbance A450 at the wavelength of 450 nm was measured.

Cells in all transfection groups were re-suspended using the complete medium (Qiagen GmbH). The kit used was the Click-iT^® EdU flow cytometry cell proliferation assay (Qiagen GmbH, Hilden, Germany). Following incubation for 120 min with 10 µM EdU at 37°C, cells were cultured for 48 h at 37°C. Following digestion with trypsin, cells were centrifuged at 1,000 × g for 5 min at 37°C and washed once with 1% bovine serum albumin (BSA) in PBS. The cells were fixed with 100 µl Click-iT fixative for 15 min at room temperature, centrifuged at 1,000 × g for 5 min at 37°C and washed once with 1% BSA in PBS. A total of 100 µl permeabilization and washing reagent (0.2%) was added for permeabilization 15 min at room temperature, then 500 µl of reaction solution containing PBS (496 ml), CuSO₄ (1 mM), Alexa Fluor 488 (10 µM), buffer additive (4 ml, component F; Qiagen GmbH, Hilden, Germany) was added to incubate for 30 min in the darkness at room temperature. Following this, 3 ml permeabilization and wash reagent was added and the mixture was centrifuged at 1,000 × g for 5 min at 37°C and washed with PBS once. The cells were then re-suspended with 500 µl permeabilization and wash reagent, and cell proliferation was detected by the Beckman Coulter FC 500 MCL/MPL flow cytometer.

Cells in all transfection groups were digested and harvested by trypsin, centrifuged for 5 min at 1,000 × g 37°C and washed once using 1% BSA PBS. Cells were re-suspended in 100 µl binding buffer (part of the apoptosis detection kit), then cells were incubated with a cell apoptosis detection kit [10 µl (15 M) Annexin V dye (FITC) and 5 µl (15 M) PI dye; Beyotime Institute of Biotechnology, Haimen, China] in the dark for 15 min at 37°C. Cell apoptosis was detected with a Beckman Coulter FC 500 MCL/MPL flow cytometer and analyzed using FCS Express 3.0 software (DeNovo software, Glendale, CA, USA).

The pNA standard was prepared according to the protocols of the Caspase-3 activity assay kit, and A405 was measured and a standard curve was plotted. Cells in all treatment groups were digested by lysis solution on ice (100 µl lysate for 5×10⁶ cells), then the supernatant was transferred to a new pre-cooled EP Tube for standby. The buffer, test article, Ac-DEVD-pNA (Qiagen GmbH, Hilden, Germany) were added to the 96-well plate successively, and incubated at 37°C for 120 min. When an evident color change was determined, the absorbance value A405 was measured immediately using a microplate reader. The absorbance value A405 reflected the activity of Caspase-3 of the test article.

MicroRNA.org online prediction indicated that the sites 3673–3690 on the 3′-untranslated region (UTR) of BMF mRNA were possible binding sites for miR-221. The DNA fragments containing the sites and containing the site mutant were synthesized in vitro, then cloned to dual luciferase gene reporter vector pMIR (Thermo Fisher Scientific, Inc.). The pMIR vector and miR-221 mimic (mimic NC, anti-miR-NC and anti-miR-221) were co-transfected into SKOV3 cells using Lipofectamine^® 2000 transfection reagent (Thermo Fisher Scientific, Inc.). Following 48 h of culture at 37°C, the luciferase activity was detected by dual luciferase assay kit. The red firefly luciferase signal was utilized as a normalization control.

Tissues or cells were lysed by radioimmunoprecipitation assay lysate (Thermo Fisher Scientific, Inc.). Supernatant was removed and the protein concentration was determined with a bicinchoninic acid assay reagent (Pierce; Thermo Fisher Scientific, Inc.). A total of 40 µg solution was loaded onto SDS-PAGE for electrophoresis separation (10% separation gel and 5% spacer gel), transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with PBS with Tween 20 (PBST, Sigma-Aldrich; Merck KGaA) containing 5% BSA for 1 h at 37°C, followed by incubation with primary antibody overnight at 4°C (the dilution ratios of BMF and β-actin were 1:2,000 and 1:4,000, respectively). The next day, the membranes were washed three times using 0.1% tris-buffered saline and Tween 20 (TBST), 5 min each time, then HRP-labeled secondary antibody (dilution ratio of 1:10,000) was added and incubated for 1 h at room temperature, then washed by 0.1% TBST ×3. The bands of PVDF membrane were developed by Supersignal West Femto HRP sensitive chemiluminescent substrate (Sigma-Aldrich; Merck KGaA), using β-actin as the internal reference substance.

Statistical analysis was performed by SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). Measurement data are expressed as mean ± standard deviation, the measurement data between the two groups were compared using Student's t-test; the measurement data between the multiple groups were compared first using one-way analysis of variance, then using the Bonferroni method for comparison. The expression levels of miR-221 and BMF mRNA in tissues were compared by Mann-Whitney U test. P<0.05 was considered to indicate a statistically significant difference.

The results of RT-qPCR demonstrated that, compared with the para-cancer tissues, the expression of miR-221 in tumor tissues was significantly increased (Mann-Whitney U=48, P<0.001; Fig. 1A), and the expression of BMF mRNA was significantly decreased in patients with ovarian cancer (Mann-Whitney U=162, P<0.001; Fig. 1B). Western blot analysis indicated that the expression of BMF proteins in ovarian cancer tissue was significantly lower than that in para-cancer tissues (P<0.001). The representative test results were depicted in Fig. 1C.

The microRNA.org online prediction indicated that BMF may be the target gene for miR-221. The predicted BMF 3′-UTR target site of miR-221 was depicted in Fig. 2A. The results of the luciferase reporter assay demonstrated that the co-transfection of the wild-type pMIR-BMF-3′-UTR-wt vector and miR-221 mimic significantly decreased the activity of luciferase (P<0.05), and the co-transfection of wild-type pMIR-BMF-3′-UTR-wt vector and anti-miR-221 significantly increased the activity of luciferase (P<0.05); however, the co-transfection of mutant pMIR-BMF-3′-UTR-mut vector and miR-221 mimic or anti-miR-221 did not significantly change the activity of luciferase, indicating a targeted regulatory association between miR-221 and BMF mRNA (Fig. 2B).

The RT-qPCR test results demonstrated that, following transfection with anti-miR-221, the expression of miR-221 in SKOV3 cells was significantly decreased (P<0.001). The transfection of si-BMF or pIRES2-BMF had no significant effect on the expression of miR-221 in SKOV3 cells (Fig. 3A). Compared with anti-miR-NC group, the expression of BMF mRNA in SKOV3 cells was increased in the anti-miR-221 transfection group. Compared with the pIRES2-blank group, the expression of BMF mRNA SKOV3 cells was significantly increased in the pIRES2-BMF transfection group (P<0.001), and the transfection of si-BMF downregulated the expression of BMF mRNA and attenuated the upregulation of anti-miR-221 on the expression of BMF proteins (Fig. 3B). Western blot analysis indicated that the transfection of anti-miR-221 or pIRES2-BMF significantly upregulated the expression of BMF protein in SKOV3 cells (P<0.001), and the expression level of BMF protein was highest in the anti-miR-221 and pIRES2-BMF combined transfection group. The transfection of si-BMF significantly inhibited the expression of BMF protein and reduced the upregulation of anti-miR-221 on the expression of BMF protein (Fig. 3C).

The EdU staining flow cytometry demonstrated that the proliferation of SKOV3 cells in the anti-miR-221 transfection group was significantly lower, compared with the anti-miR-NC group (P<0.001). Compared with the pIRES2-blank group, the proliferation of SKOV3 cells in the pIRES2-BMF transfection group was reduced significantly (P<0.001), and the transfection of pIRES2-BMF enhanced the inhibitory effect of anti-miR-221 on the proliferation of SKOV3 cells (Fig. 4A and B). The transfection of si-BMF significantly promoted cell proliferation and attenuated the inhibitory effect of anti-miR-221 on the cell proliferation (Fig. 4A and B).

Flow cytometry indicated that the apoptosis rate of SKOV3 cells in the anti-miR-221 transfection group was significantly higher, compared with the anti-miR-NC group (P<0.001). Similar to the transfection of anti-miR-221, the transfection of pIRES2-BMF significantly promoted the apoptosis of SKOV3 cells (P<0.001). The transfection of pIRES2-BMF enhanced the anti-apoptotic effect of anti-miR-221 on SKOV3 cells (Fig. 5A and B). The transfection of si-BMF significantly reduced apoptosis and attenuated the proapoptotic effect of anti-miR-221 on SKOV3 cells (Fig. 5A and B). The results of spectrophotometry demonstrated that the transfection of anti-miR-221 or pIRES2-BMF significantly upregulated the activity of caspase-3 in SKOV3 cells (P<0.001), whilst the transfection of si-BMF significantly downregulated the activity of caspase-3 in SKOV3 cells (P<0.001; Fig. 5C).