Cell culture and transfection
Ovarian cancer cells (A2780, ES-2, HO8910, CAOV3, and OVCAR3) were obtained from Cell Bank of Chinese Academy of Sciences, Shanghai, China. RPMI 1640 medium was used to maintain the cells in a humidified atmosphere of 5% CO2 at 37°C. The RPMI 1640 medium was supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin. HO8910 cells were chosen for transfection of the pcDNA3.1-FLAG-GPR176 plasmid.
Proliferation assay
Cells were seeded in 96-well plates at a density of 1,000 cells per well for 24 h. At different times (0 h, 24 h, 48 h, and 72 h), cells were incubated with 20 µL of MTT (5 mg/mL, Sigma) for 4 h. The purple formazan crystals were dissolved in 150 µL of dimethyl sulfoxide (DMSO) and the absorbance was monitored at 490 nm.
Apoptosis assay based on flow cytometry
Flow cytometry was performed with APC-labeled Annexin V (Beyotime, China) to detect phosphatidylserine externalization as an indicator of early apoptosis, and late apoptosis was detected with 7-AAD.
Wound healing assay
For the wound healing assay, 5.0 × 105 cells/well were plated into 6-well culture plates. After the cells reached 80% confluence, the cell monolayer was scraped using a tip, washed with PBS three times to remove broken cells, and grown in FBS-free medium. Cells were photographed at 0 h and 24 h after wounding. The scratch area was measured using Image software.
Cell migration and invasion assays
For the migration assay, 1.0 × 105 cells/200 µL were resuspended in serum-free RPMI 1640 and seeded in the upper chamber (BD Bioscience) and 10% FBS as a chemo-attractant was added to the lower compartment of the chamber. After incubation for 24 h, cells on the upper surface of the membrane were scrubbed, washed three time with PBS, fixed in 100% methanol, and stained with crystal violet for several minutes. For the invasive assay, the process was the same as above except a Matrigel-coated insert was used (BD Bioscience).
Patients
Ovarian cancer patients (n = 93) were recruited for surgical resection at the Affiliated Hospital of Chengde Medical University (China). Before surgery, no patient had received chemotherapy, radiotherapy, or adjuvant treatment. Prior to the start of the clinical study, all patients provided informed consent. The clinical research was approved by the ethical committee of the Affiliated Hospital of Chengde Medical University.
Tissue microarray (TMA)
Pathological specimens were fixed in 4% paraformaldehyde, dehydrated with alcohol, dealcoholized with xylene, and embedded in paraffin. Paraffin blocks were sliced into 4 µm sections and hematoxylin-and-eosin staining was used for histological analysis. Representative areas of adjacent solid tumors were identified using a microscope and corresponding tissue cores were punched out from paraffin blocks and transferred to pathological blocks with pre-made cores 4 µm in diameter.
Western blotting
Total proteins were extracted from cultured cells with RIPA lysis buffer and quantified using a BCA kit (Solarbio, PC0020). Proteins of equal volume were separated on 10% SDS-PAGE and then transferred to PVDF membranes. Nonspecific antigen sites were blocked with 5% skim milk for 1.5 h and then incubated with rabbit anti-GPR176 (1:1,000, Abcam, USA, ab122605), rabbit anti-PI3K (1:1,000, CST), rabbit anti-mTOR (1:1,000, CST), rabbit anti-p-Akt (1:1,000, CST), mouse anti-Bcl-2 (1:500, Santa Cruz), mouse anti-Bax (1:500, Santa Cruz), rabbit anti-gasdermin D (1:,5000, Abcam), rabbit anti-caspase-1 (1:2,000, Abcam), rabbit anti-E-cadherin (1:5,000, Abcam), mouse anti-N-cadherin (1:2,000, Abcam), rabbit anti-Zeb1 (1:1,000, ABclonal), rabbit anti-Snail (1:1,000, HuaBio), rabbit anti-Twist1 (1:1,000, Wanleibio), rabbit anti-VCAM1 (1:1,000, HuaBio), rabbit anti-COL4A3 (1:2,000, Abcam), mouse anti-β-actin (1:1,000, Santa Cruz), or mouse anti-GAPDH (1:2,000, Proteintech) overnight at 4℃. The membranes were washed three times and incubated with anti-rabbit or anti-mouse antibody with horseradish peroxidase (1:5,000, CST) for 2 h. Protein bands were visualized using C300 (Azure Biosystems) with the Western Bright™ ECL western blotting detection kit (Advansta, USA, K-12045-D50) and measured using Image J software (v1.8.0).
Immunohistochemistry (IHC)
The slides were deparaffinized and rehydrated three times and antigen retrieval was performed in a microwave oven for 20 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (H2O2), followed by 5% bovine serum albumin (BSA) for 30 min to block non-specific binding sites. Then, slides were incubated with rabbit anti-GPR176 (1:80, Abcam, USA, ab122605) for 3 h at room temperature. After washing three times with PBS, the slides were incubated with polyclonal swine anti-rabbit antibody with HRP (1:200, DAKO, Japan, P0399) at room temperature for 2 h. The specific binding sites were visualized with diaminobenzidine (DAB). After staining with hematoxylin, the slides were dehydrated, cleared, counted, and visualized under a microscope (Nikon, Nikon Corporation, Japan).
Immunoreactivity to GPR176 was localized in the cytoplasm. For the analysis, 100 cells were randomly chosen and counted from five representative fields by two independent researchers (YN and ZHC). The positive rate classifications were as follows: 0 = 0%, 1 = 1–49%, 2 = 50–74%, 3 ≥ 75%. The positive intensity classifications were as follows: 1 = weak, 2 = medium, 3 = strong. The immunohistochemistry (IHC) score was calculated as the intensity × positive rate, with the scores defined as follows: − = 0, + = 1–2, + + = 3–5, + + + = 6–9.
Bioinformatics analysis
The GPR176 gene expression was analyzed using the Xiantao platform (https://www.xiantao.love/), Timer (https://cistrome.shinyapps.io/timer/), and/or UALCAN (http://ualcan.path.uab.edu). The prognostic significance of GPR176 was explored using Kaplan-Meier (KM) plotter (http://kmplot.com/). In addition, differentially expressed and related genes were determined using Xiantao. The differentially expressed genes were subjected to the construction of protein-protein interaction (PPI) network and important hub genes selected. These genes were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and GSEA for the construction of signal pathways.
Statistical analysis
Statistical analysis was performed using chi-square to compare rates, Spearman correlation to analyze the rank data, Student’s t-test to compare the means, and log-rank test and Cox proportional hazards regression to conduct survival analysis. All statistical analyses were performed using SPSS 23.0 and a p-value < 0.05 was considered statistically significant.