Patient and tissue specimens
A total of 49 patients with primary invasive ovarian neoplasms who underwent curative surgery in the First Affiliated Hospital of Wenzhou Medical University from 2007 to 2015 were enrolled in this study. Two expert pathologists were invited to re-evaluate the pathological information. All these cases were classified as epithelial origin carcinomas according to the World Health Organization (WHO) classification criteria, including 32 cases of serous carcinoma (29 high-grade, 3 low-grade), 13 cases of endometrioid adenocarcinoma, 2 cases of mucinous carcinoma, and 2 cases of clear cell adenocarcinoma (Table S1). In addition, five cases of intraepithelial neoplasm and three cases of intravascular tumor emboli were enrolled. The median patient age was 56 years, ranging from 21 to 79 years. According to the 2014 Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) staging for OC, 10 of our cases were Stage I / II, while 39 cases were Stage III / IV. Stage I / II patients received a complete resection of the primary tumor, while the other patients (i.e., Stage III / IV), received cytoreductive surgery, resecting the greater omentum and both ovaries with or without the uterus. All patients received 6 to 8 cycles of chemotherapy (e.g., a taxane plus a platinum) after surgery. For the 49 patients with primary OC, clinical and follow-up data were collected for further survival analysis. The OS period was calculated from the date of the initiation surgery to the date of the patient’s death or the last follow-up visit. We also included 14 cases of benign epithelial lesions of the ovary, which served as subject controls. Informed written consents were obtained from each patient enrolled in this study. All procedures and methods in this study were ethically approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University in Zhejiang, China.
2.2. Tissue microarray construction
Paraffin-embedded tissue microarray (TMA) blocks were generated using a manual array (Beecher Instruments; Sun Prairie, Wisconsin, US). Collected 1 mm tissue cores were sampled in duplicate for each patient case, in which the typical histological areas had been marked by pathologists. Consecutive sections, 4 μm in thickness, cut from TMA blocks were prepared for immunohistochemical analysis.
2.3. Immunohistochemistry and immunostaining analysis
TMA sections were processed for immunohistochemistry (IHC) after deparaffinization and rehydration routine procedures. Antibody antigen retrieval was performed using tissue submersion in citrate buffer (10 mM, pH 6.0) with heating. Sections were then incubated with an anti-PRR11 antibody (HPA023923, Sigma-Aldrich; Saint Louis, MO, US), anti-ki-67 (cat. 27309-1-AP, proteintech; Wuhan, China), anti-N-cadherin (cat. 610920, BD Biosciences; San Jose, CA, US) and anti-early growth response protein 1 [EGR1] (ab54966, Abcam; Cambridge, UK) at 4℃ overnight. Subsequently, a two-step Envision kit (Agilent Technologies; Santa Clara, CA, US) was used to visualize positive staining. PRR11, N-cadherin, and EGR1 protein expression was evaluated by two researchers who were blind to this study using an Olympus CX31 microscope (Olympus Co; Tokyo, Japan). The antibodies were diluted with 1:100 goat serum for IHC analysis. Positivity was calculated by the semiquantitative scoring system described previously [15]. Generally, IHC staining intensity was assigned as: negative, 0; weak, 1; moderate, 2; and intense; 3. The percentage of IHC positive cells was scored as 0 to 1 (0% to100%). Theoretically, a weighted score ranging from 0 (0% of cells staining) to 3 (100% of the cells staining at 3+ intensity) was generated for each tissue core. An IHC score bigger than (>) 0 was considered positive.
2.4. PRR11 knockdown in vitro
The human OC cell lines, HO8910 and SKOV3, expressing the PRR11 protein were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). PRR11 expression was blocked via the stable transfection of a lentivirus expression plasmid that contained a small interference RNA targeting PRR11 (5’-ACGCAGGCCUUAAGGAGAATT-3’) [15]. The PRR11 knockdown plasmid was purchased from GENECHEM Corporation (Shanghai, China). An empty vector was also transfected into cells, serving as the mock control group. Knockdown efficiency of the targeted protein was evaluated by detecting the expression levels of PRR11 using Western blotting analysis.
2.5. The Trans-well migration assay
Both PRR11-knockdown cells and cells in the mock control group were harvested and introduced in the upper chamber of an 8 μm Trans-well® (Corning; Corning, NY, US) containing serum-free Dulbecco's Modified Eagle's medium (DMEM). Subsequently, 500 μl of DMEM media containing 5% fetal bovine serum (FBS) was placed into the lower chamber. The cells were then allowed to migrate for 16 hours at 37℃ and afterwards the filters between the chambers were taken out for staining using 0.5% of the crystal violet reagent. The number of cells that migrated across the filters was then manually counted under a phase-contrast microscope.
2.6. Cell proliferation assays
Cells transfected with the PRR11-siRNA or with the empty vector were seeded in 96-well plates at a density of 5,000 cells per well. The CCK-8 kit (Dojindo Laboratories; Kumamoto, Japan) was then applied to assess the cells’ short-term proliferating rate at 48 hours in vitro. The sulforhodamine B (SRB) assay was then applied to assess the long-term proliferating rate at 4-5 days in vitro.
2.7. Western blot analysis
Whole-cell lysates were prepared using a cell lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich). The protein concentration was determined using a BioRad Protein Assay Kit (BioRad Laboratories; Hercules, CA, US) according to the manufacturer’s protocol. Standard Western blotting analysis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) detected with anti-PRR11, anti-EGR1, and anti-N-cadherin antibodies using an enhanced chemiluminescence kit (Santa Cruz, CA, US). Equal protein sample loading was monitored by probing the same membrane filter with an anti-β-actin antibody. Antibodies were diluted 1:1000 in BSA.
2.8. Animal models
Cells subjected to PRR11-knockdown and control tumor cells were digested and suspended in phosphate-buffered saline [PBS] (1 × 107 cells in 0.1 mL of PBS). Then, cells were injected subcutaneously (sc) into the right flank of 4-week-old female Balb/c nude mice, respectively (each group, n=5). Two weeks after the injection, all animals were sacrificed and the tumors were resected. The tumor volume (mm3) was calculated as V = 0.52 (length × width × depth). All animal experiments were approved by the Animal Ethics Committee of the Wenzhou Medical University.
2.9. Statistical analysis
Statistical analyses were performed using the SPSS 16.0 software (IBM; Armonk, NY, US). The Chi-square (χ2) test was used for analyzing the association of categorical data. The Kaplan-Meier method was applied to estimate the OS and the log-rank test was used to assess survival differences between the groups.The data of the gene expression omnibus was from GDS3592 data set. In all these tests, a two-sided P values of less than (<) 0.05 was considered as statistically significant.