Human tissue samples and clinical data
OC tumor tissue samples were obtained from Harbin Medical University Cancer Hospital between January 2009 and December 2011 from 133 patients with OC who underwent cytoreductive surgery without any prior treatment for cancer, such as chemotherapy or radiation. The International Federation of Gynecology and Obstetrics (FIGO) staging system was applied to assess tumor stage . Histological grades were based on the World Health Organization’s Histological Grading System for tumors . Furthermore, the subjects underwent lymph node dissection to assess lymph node status. Twenty-five patients with normal ovaries who underwent hysterectomy with oophorectomy in our hospital for benign uterine disease during the same period were also included.
Approval from the Medical Ethics Committee of Harbin Medical University Cancer Hospital was obtained for the purpose of research. The 133 patients with OC were followed up for survival analyses until death or until the study closing date (January 2018).
Immunohistochemical (IHC) staining and evaluation
133 tissue blocks of OC and 25 of normal ovary tissues were cut by a microtome into 4-µm sections and affixed onto the slide. After dewaxing in xylene and rehydrating through graded alcohol concentrations, the sections were incubated with 0.3% hydrogen peroxide for 10 minutes at room temperature to block endogenous peroxidase, and then the sections were incubated with anti-LRH1 antibody (1:100, Abcam, ab223211) overnight at 4℃. After washing in phosphate-buffered saline (PBS), all sections were incubated with secondary antibodies (R&D Systems, NL004) at room temperature for 20 min and then treated with 3,3’-diaminobenzidine tetrahydrochloride (Dako, Hamburg, Germany), following by counterstaining with hematoxylin.
Protein expression level of LRH1 was scored by evaluating the percentage of the positive staining areas of tumor cells together with intensity of staining. The former was scored as follows : 0, < 0%; 1, 1-10%; 2, 11-50%; 3, 51-70% and 4, ≥ 70%. The latter was scored as follows: 0, negative staining; 1, weak staining; 2, moderate staining; and 3, intense staining. The final expression level of LRH1 was semi-quantitatively evaluated according to the sum of the scores for the percentage of positively stained tumors cells and intensity scores (0-7) in which the final staining scores of 0-3 and 4-7 were considered to be low and high expression, respectively.
The IHC staining on each slide was scored in twice independently by two pathologists who were sophisticated in evaluating IHC and blinded with the clinicopathological information.
The human OC cell lines SKOV3 and OVCAR3 (ATCC) were employed. Both the cell lines have been approved by short tandem repeat (STR) profiling. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 in DMEM complemented with 10% Fetal bovine serum (FBS) (Hyclone, USA) and antibiotics (penicillin and streptomycin). Cells were passaged when they reached 80% confluence.
Western blotting analysis
The cell lysates were both centrifuged at 13000 rpm (at 4℃ for 5 minutes). After that 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) were used to segregate them, and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA).The filters were blocked with blocking buffer (Sangon Biotech) for 45min.Membranes were incubated with Primary antibodies anti-LRH1 (Abcam, ab223211), anti-E-cadherin (Abcam, ab1416), anti-N-cadherin (Abcam, ab18203), anti-Vimentin (Abcam, ab92547), anti-β-catenin (Cell Signaling Technology, D10A8)) at 4℃ overnight, and with secondary antibodies at 1:5000 dilution at room temperature for 2h, following by incubating with β-actin (Abcam, ab179467) atroom temperature for 1.5h.
RNA extraction and reverse transcription (RT)-qPCR
The TRIzol™ LS Reagent (Invitrogen™) was used to extract total RNA from tissues and cells, according to the manufacturer's protocol, followed by complementary DNA synthesis using a PrimeScript RT reagent Kit with gDNA Eraser (Takara). Amplification was subsequently carried out using the GeneAmp® PCR System 9700 (Applied Biosystems, USA). Real-time PCR was performed using LightCycler® 480 Ⅱ Real-time PCR Instrument (Roche, Swiss). The mRNA expression levels of LRH-1 were normalized to the mRNA levels of GAPDH, which was used as an internal control. The 2-ΔΔCt method was used to quantify the mRNA expression levels .
Establishment of stable short hairpin (sh)RNA‑mediated LRH‑1 knockdown ovarian cancer cell lines.
shRNA-induced knockdown of LRH-1 expression was achieved using the lentiviral expression system. The plasmid used in the present study were pPLK/GFP+Puro-NR5A2 (Genomeditech, China). Viral particles were generated by co-transfecting 293T cells (ATCC) with the shRNAs and the GM easyTM Lentivirus Packaging kit (Genomeditech, China), which contains packaging plasmids and a transfection reagent according to the manufacturer's protocol. Subsequently, the shRNA viral particles transfected SKOV3 and OVCAR3 cells with 4 μg/mL polybrene (Sigma-Aldrich), and stable cell lines were established after 10 days of puromycin (2μg/ml) selection. Knockdown was confirmed using RT-qPCR or immunoblotting. The selected cell lines were routinely cultured in puromycin-containing media until 2 days prior to experimentation.
For migration assays, infected OVCAR3 cells (1×105 in 200 μL of serum-free DMEM medium) and SKOV3 cells (1×105 in 200 μL of serum-free DMEM medium) were seeded into the upper chamber of transwell plates in a 24-well format with 8 μm diameters (Corning Costar, USA). Then, 600 μL of medium containing 10% FBS was added to the bottom chamber as a chemoattractant. After 24 hours of culture, cells were fixed with methanol and stained with Crystal Violet solution. The remaining cells were removed from the top of the permeable membrane using a cotton swab. Then, cells that migrated through the upper chamber were counted in 4 random fields under a light microscope (NIKON INSTRUMENTS（SHANGHAI）CO.,LTD).
Wound healing assay
OVCAR3 (1×105 cells) and SKOV3 cells (1×106 cells) were seeded in 6-well cell culture plates and incubated for 24 hours at 37℃. After achieving confluence, the cellular layer in each plate was scratched using a plastic pipette tip. The migration of the cells at the edge of the scratch was analyzed at 0 and 24 hours, when microscopic images of the cells were captured.
Cell counting kit-8 (CCK-8)
SKOV3 and OVCAR3 cells were plated in flat-bottom 96-well plates (1500 and 1000 cells/well) and supplemented with 100μL DMEM medium with 10%FBS per well. After incubation at 37°C in a humidified incubator with 5% CO2 for 2, 24, 48, 72, and 96 hours, respectively, 10 μL of CCK-8 (Yeasen Biotech Co., Ltd.) was added to each well. Then, after 1 hours of culture, colorimetric analysis was performed on a microplate reader (Thermo Scientific™ Varioskan™ LUX) at a wavelength of 450 nm. The assay was performed using six replicates.
The chi-square test was used to analyze the statistical differences of the clinicopathologic variables. Overall survival (OS) and disease-free survival (DFS) were evaluated by the Kaplan-Meier method and log-rank test. The Cox proportional hazards model was used to estimate the independent prognostic factors for survival. Logistic regression was performed for multivariate analysis of the association between LRH1 expression and intraperitoneal metastasis. Student's t-test were used to analyze the differences between the experimental and control groups. A two-sided P < 0.05 was considered significant.
UALCAN (http://ualcan.path.uab.edu) is an interactive web resource based on TCGA database, which can be used to analyze relative transcriptional expression of potential genes of interest between tumor and normal samples and association of the transcriptional expression with relative clinicopathologic parameters . In this study, we use UALCAN to explore the association of LRH1 mRNA expressions in ovarian cancer tissues and clinicopathologic parameters. Students’ t test was applied to compared the difference of mRNA expression and p <0.01 was considered as statically significant.
GEPIA (http://gepia.cancer-pku.cn/) is a web server to analyze cancer and normal gene expression and interaction . In this study, we use GEPIA to obtain a series of genes that have similar expression patterns with LRH1 in ovarian cancer.
The Metascape (http://metascape.org/) is an online analytical tool, which facilitates gene annotation integration, functional enrichment, interactome analysis . In the present study, Metascape was applied to gene-enrichment analysis of genes that have similar expression patterns with LRH1 in ovarian cancer.