Experiments were approved by the Institutional Review Board of Shengjing Hospital at China Medical University. A total of 45 samples were collected from tissues removed during the surgical removal of cervical cancers at the Department of Gynecology, Shengjing Hospital, China. The 45 samples comprised 30 samples of ovarian epithelial cancer tissues and 15 samples of normal ovarian tissues. All the tissues were removed during surgery whereupon they were immediately frozen in liquid nitrogen and stored at −80°C. Based on the characteristics of ovarian carcinoma, it is extremely difficult to obtain para-carcinoma tissue; therefore, normal ovarian tissues were used as a control group. Patients anonymity was maintained. The tissue samples were examined by specialists to obtain a final diagnosis, and no patients were administered chemotherapy or radiotherapy prior to surgery. The clinicopathological characteristics of 30 patients of epithelial ovarian cancer are shown in Table I.

In total, 45 fresh tissue samples were embedded into paraffin sections and an additional 136 different paraffin slices were obtained from the ovarian tissues resected during operations at the Department of Gynecology, Shengjing Hospital, China Medical University, China, from 2008 to 2012. A total of 181 paraffin sections were thus obtained: 112 of primary malignant ovarian tumors, 23 of borderline ovarian tumors, 26 of benign ovarian tumors and 20 of normal ovarian tissues. The tissue sections were examined by experienced specialists to obtain a final diagnosis. Histopathological diagnoses were made using the World Health Organization criteria. The classification of cancer stage and grade was carried out according to the International Federation of Gynecology and Obstetrics. The clinical and pathological information concerning the patients was collected from their clinical records, including their age, surgical stage, lymph node metastasis, pathological tumor grade and subtype, and residual tumor size.

The age range (median) was 16–77 years (52.7 years) in the malignant ovarian tumor group; 21–78 years (41.5 years) in the borderline ovarian tumor group; 24–61 years (43.2 years) in the benign ovarian tumor group; and 45–68 years (58.9 years) in the normal ovarian tissue group. There were no statistically significant differences in the ages of these groups (P>0.05).

Information was collected on the clinical chemotherapeutic treatments received and the follow-up from 77 patients out of a total 112 patients with malignant ovarian cancer (the information from 77 patients was complete and these 77 patients were followed-up for 24 months at least after surgery). These 77 patients underwent treatments for ovarian cancer that included surgical debulking followed by 6–8 postoperative cycles of conventional chemotherapy, consisting of paclitaxel (Yangtze River Pharmaceutical Group, Taizhou, China) and carboplatin (Qilu Pharmaceutical Co., Ltd, Jinan, China). Overall survival (OS) was defined as the date of surgery to the date of death or the last follow-up. Disease-free survival (DFS) was defined as the interval from the initial surgery to clinically or radiologically proven recurrence/metastasis and deceased. Following surgery, the patients were observed at 3-month intervals. As of May 2014, it has been 70 months since the first patient was recruited into the research group, and it has been 24 months since the last patient was recruited into our group. The median followup period was 48 months. To determine the factors influencing survival after surgery and standard chemotherapy, conventional variables, together with hMOF expression, were assessed in 77 ovarian carcinoma patients.

The PrimeScript™ 1st Strand cDNA Synthesis and PCR amplification kits were purchased from Takara (6110A, RR02A; Dalian, China). Mouse monoclonal anti-hMOF antibody (GTX83065) was obtained from GeneTex (Irvine, CA, USA). The anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) monoclonal antibody was purchased from Boshide Biotech (BM1985; Wuhan, China).

Total RNA from epithelial ovarian cancer and the normal ovarian tissues were isolated using TRIzol^® LS reagent (Invitrogen, Carlsbad, CA, USA). RNA (500 ng) from each sample was used as a template to produce cDNA using the PrimeScript™ 1st Strand cDNA Synthesis kit. PCR reactions were performed under the following conditions: an initial denaturation step at 95°C for 2 min, followed by 30 cycles of denaturation step at 95°C for 30 sec, annealing at 57°C for 30 sec and extension at 72°C for 30 sec. The primer sets used for the PCR were: GAPDH, forward: 5′-ATCACTGCCACCCAGAAGAC-3′ and reverse: 5′-ATGAGGTCCACCACCCTGTT-3′, yielding a 433-bp product; hMOF, forward: 5′-GACACTGTACTTTGACGTGGAGC-3′ and reverse: 5′-CACTGTGATGGGTGGTTTCTT-3′, yielding a 493-bp product.

Soluble proteins were isolated from tissues for western blotting. The protein concentrations were measured by bicinchoninic acid (23228; Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein from each sample were separated by electrophoresis on an SDS-10% polyacrylamide gel, transferred to a polyvinylidene difluoride membranes, and blocked with 5% non-fat dry milk in 1X TBS plus 0.1% Tween-20 at room temperature for 2 h. The membranes were incubated overnight at 4°C with primary antibodies in 1% bovine serum albumin in 1X TBS plus 0.1% Tween-20. The primary anti-hMOF monoclonal antibody (diluted 1:1,000) was purchased from GeneTex (GTX83065) and the anti-GAPDH monoclonal antibody (diluted 1:2,000) was purchased from Boshide Biotech (BM1985). Membranes were washed and incubated again for 2 h at room temperature with horseradish-peroxidase-conjugated anti-mouse secondary antibodies. Proteins were visualized with ECL reagent (ECL Prime Western Blotting Detection Reagent; Amersham, Pittsburgh, PA, USA). The experiments were repeated three times.

Paraffin-embedded histological sections from each group of ovarian tissues were cut into 5-μm slices. Immunohistochemistry was used to analyze the hMOF protein expression levels. Mouse monoclonal anti-hMOF antibody (GTX83065; diluted 1:800) was purchased from the GeneTex. The staining procedure was performed according to the manual of an ultrasensitive streptavidin-peroxidase kit (KIT-9701; Maixin Bio, Fuzhou, China) and Harris’s hematoxylin was used to stain the cell nuclei. Tissues were treated with phosphate-buffered saline instead of primary antibody as a negative control. Buff-colored granules in the cell nucleus were considered a positive result. The tissues were rated according to their chromatic intensity: no pigmentation, 0; light yellow, 1; buff, 2; and brown, 3. Five high-power fields in serial sections from each slice were selected, scored, and the mean percentage of chromatic cells was estimated: <5% chromatic cells, 0; 5–25% chromatic cells, 1; 26–50% chromatic cells, 2; 51–75% chromatic cells, 3; and >75% chromatic cells, 4. The two numbers (intensity score × percentage chromatic cells) were multiplied and scored: 0–2 was considered (−), 3–4 (+), 5–8 (++) and 9–12 (+++). The 112 patients of ovarian cancer were divided into the high hMOF expression group (++/+++) and the low hMOF expression group (−/+). Two observers read the sections to control for systemic error.

The gene expression and western blot images were scanned and quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA). Differences in proportions were evaluated using the χ² test. The χ² test or Fisher’s exact test, whichever was appropriate, was used to analyze the relationship between hMOF expression and clinicopathological variables. A survival curve was generated using the Kaplan-Meier method and compared by the log-rank test. Cox’s proportional hazard regression model was used for multivariate survival analysis of prognostic factors. Statistical analyses were performed using SPSS v17.0 software (SPSS, Inc., Chicago, IL, USA). The Student’s two-tailed t-test was used in all analyses. P<0.05 was considered to indicate a statistically significant result.

hMOF plays a role in the basic physiological processes of mammalian cells and therefore, it may be involved in different malignant tumors. Consequently, we hypothesized that hMOF may be important in the occurrence and development of ovarian cancer and that the role of hMOF may differ between ovarian cancer and normal ovarian tissues. RT-PCR showed that the expression of hMOF mRNA in normal ovarian tissues (15 cases) was 2.62-fold higher than that in ovarian cancer tissues (30 cases) (P<0.05; Fig. 1A and B).

The results from the western blot analysis and immunohistochemical methods used to investigate hMOF protein expression in different ovarian tissues, confirmed the results from the mRNA expression analysis. Western blot analysis showed that hMOF protein expression in the normal ovarian tissues was 1.28-fold higher than that in ovarian cancer tissues (P<0.05; Fig. 2A and B).

hMOF immunoreactivity was identified in the nucleus (Fig. 3) and in all tissue types, and hMOF was expressed in the nucleus. The positive hMOF protein expression rates in ovarian epithelial cancer, borderline tumor, benign tumor and normal ovarian tissues were 80.36, 65.22, 84.62 and 80.00%, respectively (Table II). The positive rates did not significantly differ between groups. Data were divided into a low hMOF expression group (including negative and weak positive expression) and a high hMOF expression group (including moderate and strong positive expression). According to the standard scoring of paraffin slices, 3–4 was defined as weak expression and 5–8 was defined as moderate expression. The specific scoring criteria was as described in Materials and methods. Further analysis of the data found that the proportions of high hMOF expression in ovarian epithelial cancer, borderline tumor, benign tumor and normal ovarian tissues, were 27.68, 30.43, 57.69 and 50.00%, respectively (Table II). Therefore, hMOF protein expression in ovarian benign tumor tissues and normal ovarian tissues was much higher than that in ovarian epithelial cancer tissues (P<0.05).

A total of 112 ovarian cancer patients were divided into the high (++/+++) and low (−/+) hMOF protein-expression groups. The rate of high hMOF protein expression in stage I ovarian cancer tissues was 33.33%, significantly higher than its expression in stage IV ovarian cancer tissues (P<0.05). The protein expression of hMOF in ovarian endometrioid adenocarcinoma tissues was significantly higher than its expression in ovarian serous adenocarcinoma tissues (P<0.05). There was no relationship between hMOF protein expression with cell differentiation and lymph node metastasis (P>0.05; Table III).

A total of 77 patients with complete follow-up data were divided into the low (−/+) and high (++/+++) hMOF protein-expression groups. The COX proportional-hazards regression model was applied with survival time as an independent variable and dependent variables including the age of ovarian cancer patients, surgical pathological staging, differentiation, pathological category, lymph-node metastasis, residual tumor size and hMOF expression. Multivariate analysis revealed that the age of ovarian cancer patients and hMOF expression were independent prognostic risk factors for OS (Table IV), while residual tumor size and hMOF expression were the independent risk factors closely associated with DFS (Table IV).

As of May 2014 (70 months since the recruitment of the first patient and 24 months since recruitment of the last patient), 26 patients in the low hMOF-expression group (comprising 53 patients) succumbed, while five patients in the high hMOF-expression group (comprising 24 patients) succumbed. A Kaplan-Meier analysis (Fig. 4) showed that the OS and DFS rates were significantly higher in the high hMOF expression group compared to the low hMOF expression group (log-rank test; OS, P=0.018 and DFS, P=0.009).