The A2780 (#CL-0013) and SKOV3 (#CL-0215) ovarian cancer cell lines were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China). HO-8910 was from our laboratory. A lentiviral vector was used to overexpress LPA3 in A2780, HO-8910 and SKOV3 cell lines. The reagents that contain LPA (C18:1, #857230P), epidermal growth factor (EGF, #SRP3027) and pertussis toxin (PTX, #P7208) were purchased from Sigma-Aldrich. Other reagents were obtained from Selleck Chemicals, such as PD98059 (#S1177), SP600125 (#S1460), BAY11-7082 (#S2913), AG1478 (#S2728) and SB203580 (#S1076). The anti-p-EGFR (#3777), anti-EGFR (#2085), anti-p-ERK1/2 (#9101), anti-ERK1/2 (#4695), anti-p-JNK (#4671), anti-JNK (#9252), anti-p38 (#8690), anti-p-p38 (#4511), anti-Akt (#4691), anti-p-Akt (#4060), anti- NF-κB (#8242), anti-p-NF-κB (#3033) and anti-α-tubulin (#3873) were obtained from Cell Signaling Technology.
The gene expression data of normal ovarian tissue were extracted from Genotype-Tissue Expression (GTEx) database. The gene expression data of ovarian cancer tissue were obtained from The Cancer Genome Atlas (TCGA) database. Then differential gene expression analysis was performed by R Language in ovarian cancer samples and normal ovarian samples.
Cell viability assay
When cells were attached to bottom of 96-well plates, changed to starvation medium for overnight. The cells were stimulated with EGF (20 ng/ml), PD98059 (5 µM), LPA (1 µM), Ki16425(1 µM), SB203580 (1 µM), PTX (100 ng/ml), BAY11-7082 (1 µM) and SP600125 (5 µM) for 24 h. The activity of cell proliferation was detected by TransDetect TM Cell Counting Kit (CCK, # FC101-01, TransGen Biotech).
Cell migration assay
Cell migration activity was measured by 24-well transwell chambers assay (Transwell, Corning Costar). The cell culture insert with an 8-µm pore size were placed in 24-well plates (lower chamber) containing 500 µl of starvation medium with various stimulants, including LPA (1µM), Ki16425 (1 µM), EGF (20 ng/ml), SB203580 (1 µM), PD98059 (5 µM), SP600125 (10 µM), BAY11-7082 (1 µM), and AG1478 (1 µM). Then, cells were placed in the cell culture insert with 200 ul of starvation medium (upper chamber). The 24-well plate was returned to the cell incubator for 6 h. The cells were pretreated with PTX (100 ng/ml) for 24 h and then carried out the cell migration assay.
The male BALB/c nude mice at 4 weeks of age were purchased from Charles River Laboratories (Beijing, China). Tumor xenograft mice model were implemented by stably overexpressing LPA3 in A2780, HO-8910 and SKOV3 cells. Each BALB/c nude mouse was injected with 4 × 106 control group cells and experimental group cells at its the right and left flanks, respectively. Each kind of ovarian cancer cells were inoculated into 5–6 mice. The tumor size was measured by vernier caliper weekly until the animals were killed 30 days later. Then the xenograft tumors were subjected to qPCR and immunohistochemistry analyses.
We established a lung metastases model of ovarian cancer in nude mice to evaluate the effects of LPA3 on tumor metastasis. For the intravenous injection, the cancer cells (2×106) were injected via the caudal vein to induce lung metastasis. After 30 days, Luciferin (Promega) was injected intraperitoneally into mice at a dose of 150 mg/kg, and within 30 min after injection, luciferase imaging was performed. Then the mice were killed and counted the number of lung metastatic nodules which were verified by hematoxylin-eosin (HE) staining.
HE staining and immunohistochemistry
The tumor tissues were dewaxed and stained with an HE staining kit (#G1120, Solarbio, China). The pathological changes were observed under a microscope. The 4-µm thick sections slides were cut from the paraffin blocks of tumor tissues, then deparaffinized for 15 min after drying. The slides were incubated with primary antibodies for LPA3 (#bs-2882R, BIOSS) and Ki-67 (#bs-23103R, BIOSS) overnight at 4°C after antigen retrieval and serum closure. The slides were incubated with secondary antibodies for 60 min. The slides were stained with DAB and HE, then detected by a Zeiss microscope.
The other methods
The western blot, tissue microarrays and qPCR were performed as described previously[5, 16]. The cAMP was measured by GloSensor™ cAMP Assay (Promega). Tissue arrays of human ovarian cancer were purchased from Shanghai Outdo Biotech. The F-actin of ovarian cancer cells were stained with phalloidin-Alexa Fluor 555 (#C2203S) obtained from Beyotime.
All experimental data were performed as mean ± S.E.M. from at least three independent experiments by GraphPad Prism 7.0 software analysis. Differences with P < 0.05 were considered statistically significant.