Cell lines and culture conditions
The human epithelial ovarian cancer cell lines HEY, OV-90 and HO-8910 were purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai, China. The HEY cells and A2780 cells were cultured in DMEM medium and RPMI-1640 medium respectively with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The HO-8910 cells were cultured exactly like A2780 cells. The OV-90 cells were cultured in half MCDB 105 medium and half 199 medium with 15% bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were cultured at 37 °C in 5% CO2 and 95% air.
Chemical inhibitors and antibodies
The chemical Kira6 (IRE1α kinase inhibitor) was purchased from Selleck, TX, USA. The antibodies ROR2, caspase3, caspase7, PARP, cleaved caspase3, cleaved caspase7, cleaved PARP, BIP, CHOP, IRE1α, c-Jun, phosphorylated IRE1α, phosphorylated JNK(Thr183/Tyr185), phosphorylated c-Jun(Ser73) and β-actin used for western-blot assay were purchased from Cell Signaling Technology, MA, USA. The antibodies Bcl-2, Bax and JNK used for western blot assay were purchased from Santa Cruz, CA, USA. The antibody phosphorylated IRE1α(Ser724) used for western-blot assay was purchased from Abcam, Cambridge, UK. The antibodies ROR2, BIP and CHOP used for immunohistochemistry were purchased from Abcam, Cambridge, UK.
Tissue samples for western-blot assay
Tissue samples were collected from 23 HGSOC patients who underwent surgical resection at Qilu Hospital of Shandong University in first half of 2019. The ages ranged from 41 to 80 (median: 62 years). 21.8% patients were diagnosed at early stages (FIGO stage I–II) and the others were diagnosed at advanced stages (FIGO stage III–IV). All patients were diagnosed based on clinical protocols without previous neo-adjuvant chemotherapy or immunotherapy. Meanwhile, fimbriae of the fallopian tubes were collected from 23 patients receiving bilateral salpingectomy with benign neoplasms at the same hospital as normal control tissues.
GEO data sets analysis
Gene expression profiles of GSE69428, GSE40595 and GSE18520 were downloaded from Gene Expression Omnibus (GEO). Dataset GSE69428 included HGSOC and paired normal FTE samples from 10 independent patients. Dataset GSE40595 included epithelial tumor samples from 32 HGSOC patients and 6 normal OSE samples. Dataset GSE18520 included 53 HGSOC tissue samples and 10 normal OSE samples. All the samples were isolated laser based microdissection using the Affymetrix human genome U133 Plus 2.0 microarray. The “limma” package was used to analyze differentially expressed genes (DEGs) between HGSOC and normal FTE or OSE samples. The adjusted P < 0.05 and |log2fold change (FC)| > 1 were set as the cut-off criteria. Adjusted value (adj. P) was applied to correct false-positives.
Adenovirus transfection and chemical treatment
The adenovirus was purchased from Vigene Bioscience in Jinan, China. Cells were suspended with normal culture medium and seeded in 6-well plate at 20 × 104 cells/well overnight to adhere. Volume of adenovirus was measured according to the MOI (volume = [cell number × MOI]/virus titer, MOIHEY = 50, MOIOV-90 = 50, MOIHO-8910 = 30). After transfected in opti-MEM with adenovirus for 6 h, normal culture medium was replaced for the following cultural. To examine the effects of IRE1α kinase inhibitor on apoptosis and relevant molecules, HEY and HO-8910 cells were first infected with ROR2 overexpression or negative control adenovirus for 6 h and then treated with 2 μM Kira6 for 72 h before collecting for the following assays.
The patient tissue chip was purchased from Alenabio, Xian, China. Fresh tissues from tumor xenograft were fixed with 4% paraformaldehyde for 24 h before dehydrated and embedded in paraffin. Tissue sections were torrefied for 1 h and dewaxed with xylene and ethyl alcohol. The microwave antigen retrieval technique was used to repair the antigen. Sections were incubated with 3% H2O2 for 20 min to block endogenous peroxidase activity and incubated with goat serum for 30 min to block non-specific antigens. Then sections were incubated with primary antibodies (ROR2, 1:400, BIP, 1:300, CHOP: 1:300) overnight at 4 °C. After incubated with biotin-labeled goat anti-rabbit IgG polymer and horseradish enzyme-labeled streptomycin for 30 min, respectively, positive signals were detected with DAB reagent and quantified by Image-Pro Plus software 6.0 (Media Cybernetics, USA). The same setting was used for all the analyzed tissues to be accurate for the staining reading. Integrated optical density (IOD) and size of the total area was measured in each field, and staining score was formulated as IOD/size.
Cells were washed with 1× PBS for 3 times and cells lysates were prepared in RIPA lysis buffer with 1% phenylmethanesulfonyl fluoride (PMSF) and 1% sodium fluoride. Protein concentrations were detected with BCA protein assay kit (Beyotime, Beijing, China). Total proteins were separated on a 12% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. Membranes were blocked with 5% defatted milk for 1 h at room temperature and incubated with the primary antibodies overnight at 4 °C. Then membranes were washed with 1× TBS and incubated with appropriate secondary antibodies. Bands were detected using chemiluminescent substrate (Thermo Fisher Scientific Inc., MA, USA) and quantified by ImageJ software (National Institutes of Health, USA). The β-actin band was served as control.
Quantitative real-time transcription-polymerase chain reaction
Total RNA of cells was extracted and concentration and purity were detected using spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). Then the RNA (3000 ng/20 μl reaction system) was transcribed into cDNA. PCR reaction was performed on StepOne™ PCR amplifier (Applied Biosystems, USA) with SYBR-green (TAKARA, Japan) in a 10 μl reaction system, and β-actin was used as the control. Primers for human ROR2 gene were as follows: forward: 5′-GTGCGGTGGCTAAAGAATGAT-3′, reverse: 5′-ATTCGCAGTCGTGAACCATATT-3′. Relative gene expression levels were normalized to β-actin. Primers for β-actin gene were as follows: forward: 5′-CTCACCATGGATGATGATATCGC-3′, reverse: 5′-AGGAATCCTTCTGACCCATGC-3′.
After transfected with negative control or ROR2 adenovirus, cells were suspended at respective concentrations (HEY 1500 cells/100 μl, OV-90 2500 cells/100 μl, HO-8910 2000 cells/100 μl) and seeded in 96-well plates (100 μl/well) overnight to adhere. 10 μl MTT solution (5 mg/ml) was added into every well at fixed time from Day1 to Day6. After incubated at 37 °C for 4 h, supernatant liquor was discarded and 100 μl DMSO was added in every well to dissolve the formazan. Absorbance was read at 490 nm using a microplate reader (Tecan Group Ltd., Männedorf, Switzerland).
Colony formation assay
After transfected with negative control or ROR2 adenovirus for 24 h, cells were suspended at respective concentrations (HEY 500 cells/2 ml, OV-90 1000 cells/2 ml, HO-8910 500 cells/2 ml) and seeded in 6-well plate (2 ml/well). After incubated at 37 °C for 12 days, cells were fixed with 4% paraformaldehyde for 5 min and stained with crystal violet (Beyotime, Beijing, China) for 30 min.
Cells transfected with negative control or ROR2 adenovirus for 72 h were suspended at respective concentrations (HEY 5 × 104 cells/200 μl for invasion and 3 × 104 cells/200 μl for migration, HO-8910 8 × 104 cells/200 μl for invasion and 5 × 104 cells/200 μl for migration) and seeded in transwell chambers (8 μm pore size; Corning Costar, MA, USA) with or without Matrigel (60 μl, 1:9 dilution in serum free medium, BD Biosciences, CA, USA). After incubated at 37 °C for 24 h, cells were fixed with 4% paraformaldehyde for 5 min and stained with crystal violet (Beyotime, Beijing, China) for 30 min. Images were taken with JEM-1200 EX II Electron Microscope (JEOL, Tokyo, Japan).
Flow cytometry assay
Flow cytometry assay was used to detect the apoptotic ratio of HGSOC cells. After transfected with negative control or ROR2 adenovirus for 72 h, cells were digested with tyrisin without EDTA and suspended at 1 × 106 cells/ml. Cells (100 μl/tube) were dyed with 5 μl fluorescein isothiocyanate (FITC) Annexin-V and PI and 5 μl propidium iodide (PI) (BD, NJ, USA). After incubated at room temperature for 15 min, 400 μl binding buffer was added into each tube and cells were collected with flow cytometry (BD Biosciences, San Jose, CA, USA). Quantitative analysis of apoptotic ratio was performed by CellQuest Pro software (BD Biosciences, Franklin Lakes, NJ, USA).
RNA sequencing assay
HO-8910 cells were seeded in culture dish at 1.5 × 106 cells/dish (55 cm2) overnight to adhere. Volume of ROR2 overexpression and negative control adenovirus were calculated with MOI of 30. After transfected for 6 h, cells were changed with normal medium. After 48 h, cells were disrupted with Trizol Reagent (Thermo Fisher Scientific Inc., MA, USA) and sent to Novogene Corporation (Beijing, China) for whole transcriptome sequencing. The libraries were sequenced using the IlluminaHiSeq™ 4000 sequencing platform.
Plasmid extraction, siRNA and transfection assay
The plasmid was purchased from Genechem, Shanghai, China. 1 μg plasmid was transfected into competent cells. Shaking flask culture with 100 μg/ml ampicillin was used for amplification of bacteria. Then plasmids were extracted with plasmid extraction kit (Omega, GA, USA) and the concentration and purity were measured with spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). Lentivirus expressing ROR2 packaged with psPAX2 (Addgene, MA, USA) and pMD2G (Addgene, MA, USA) were produced in HEK293T cells with Lipofectamine 2000 (Invitrogen, CA, USA). Stable cells were selected for 10 days in medium with 4 μg/ml puromycin (Solarbio, Beijing, China) after transfection by Lentivirus for 12 h. The small interfering RNA (siRNA) targeting IRE1α was synthesized by BioSune (BioSune, Shanghai, China). Cell transfection was performed using Lipofectamine 2000 (Invitrogen, CA, USA).
Tumor xenograft experiment
To verify the effects of ROR2 on ovarian cancer in vivo, ROR2 stable overexpression cells were constructed with Lentivirus PCMV-ROR2. Cells transfected with PCMV-NC were used as control. 1 × 107 cells in 100 μl PBS were injected subcutaneously into either side of the armpit of the same 4-week-old nude female mice. Tumor sizes were measured every other day 10 days after injection. Sizes of tumors were measured (Volume = [length × width2]/2) with vernier caliper every other day. Mice were sacrificed 33 days after injection and tumors were removed and weighted. Part of the tumors were fixed with 4% paraformaldehyde for immunohistochemistry and the others were used for western-blot assay. The research was approved by the Experimental Animal Ethics Committee of Qilu Hospital of Shandong University (Approval number: KYLL-2016-338).
All experiments were repeated three times at least. Data were analyzed with Mann–Whitney test, Student’s t test or 2way Analysis of Variance (ANOVA) test and shown as mean ± standard error of mean (SEM). Statistical significance was defined as P < 0.05. All the statistical analysis was performed using GraphPad Prism Version7.00 (GraphPad Software, USA).