Reagents and antibodies
Antibodies against SphK2, c-Myc and GAPDH were purchased from Abcam. Antibodies against ERK1/2, phosphor-ERK1 (Thr202/Tyr204)/ERK2 (Thr185/Tyr187), cyclin D1, phosphor-Rb were purchased from Cell Signaling Technology. U0126 was ordered from Sigma-Aldrich.
Tissue specimens were collected from the patients without preoperative chemotherapy, including 5 normal ovarian tissues and 50 primary epithelial ovarian cancer (PEOC) tissues (stage I-II 20 cases, stage III-IV 30 cases). All patients signed the informed consent approved by Institutional Review Board of Shanghai Jiaotong University.
Cell lines and culture conditions
Human EOC cell line SKOV3 and OVCAR3 were obtained from American Type Culture Collection. SKOV3 was routinely cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 1% antibiotics. OVCAR3 was routinely cultured in RPMI-1640 medium (Invitrogen) supplemented with 20% fetal bovine serum, 0.01 mg/ml insulin (Sigma-Aldrich) and 1% antibiotics. A2780 was purchased from the China Center for Type Culture Collection and routinely cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotics. Immortalized ovarian surface epithelial (IOSE) cell line was a gift from Prof. MW-Y Chan (National Chung Cheng University, Taiwan) and cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotics.
Immunostaining was performed and scored as previously described . The primary antibody used was anti-SphK2 (1:100, Abcam). Staining index (SI) was calculated as staining intensity score × proportion score. The protein expression level was considered to be high when score was >3, and low when score was ≤3.
Western blot analysis
Cells were harvested after the indicated treatments. The protein concentration was determined using BCA reagent. Western blotting was performed as previously described .
Transient (siRNA) and stable (shRNA) transfection
The siRNAs specifically targeting human ERK1 (5'-CAUGAAGGCCCGAAACUACUU-3'), ERK2 (5'-GCGCUUCAGACAUGAGAACUU-3'), and the scrambled control siRNA (5'-AAUUCUCCGAACGUGUCACGU-3') were synthesized by GenePharma. siRNA duplexes were transfected using Lipofectamine (Invitrogen) according to the manufacturer’s protocol. The shRNA lentiviral packaging plasmid specifically targeting human SphK2 (5'-AACCUCAUCCAGACAGAACGA-3') and the non-targeting negative control plasmid (5'-AAUUCUCCGAACGUGUCACGU-3') were constructed by GenePharma. Cell lines were transduced with lentiviral vectors at multiplicity of infection (MOI) = 5. To establish the cells stably down-regulating SphK2, transfected cells were selected by culturing in puromycin. Single colonies of stable transfectants were isolated and expanded.
RNA was extracted by TRIzol Reagent. The mRNA levels were measured by SYBR Green RT-PCR and then calculated by 2-ΔΔCt method. Primers were as follows: SphK2, 5'-GGTTGCTTCTATTGGTCAATCC-3' (forward) and 5'-GTTCTGTCGTTCTGTCTGGATG-3' (reverse); GAPDH, 5'-TGCACCACCAACTGCTTAGC-3' (forward) and 5'-GGCATGGACTGTGGTCATGAG-3' (reverse) .
Cellular proliferation assay
Cell proliferation was assessed using WST-1 (Roche) assay as previously described . Briefly, the indicated cells were seeded into 96-well plates and the cell number was measured at 24, 48, 72 and 96 h. At each time point, WST-1 assay reagent was added into each well and cultured at 37℃ for 2 h. The supernatant from each well was then collected for measurement of absorbance at 450 nm. There is a direct correlation between the cell number and the absorbance at 450 nm in the current study.
The animal experiments were performed according to the Laboratory Animal Guidelines provided by Shanghai Jiao Tong University School of Medicine. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine. All the experiments and methods were performed in accordance with the relevant guidelines and regulations. Mice were purchased from the Chinese Academy of Sciences. To establish subcutaneous transplantation models, female BALB/c nu/nu mice aged 5-6 weeks (8 mice in each group) were subcutaneously injected with 2× 106 EOC cells stably transfected with indicated plasmids. Tumor volumes were calculated twice a week using the following formula: V = (largest diameter × small diameter × depth) ×p /6. 28 days after injection of EOC cells, the mice were sacrificed and the weight of tumors was measured.
Cell cycle analysis
Cells were trypsinized, fixed in 70% ice-cold ethanol overnight, and stained with propidium iodide at room temperature for 45 minutes. The cellular DNA content was analyzed by flow cytometry.
The Annexin V-FITC/PI apoptosis detection kit (BD, USA) was used to identify apoptotic cells by flow cytometry following the manufacturer's instructions.
Tumor RNA was prepared with RNeasy Plus Mini Kit (Qiagen) according to manufacturer’s protocol. Total RNA was subjected to microarray analysis with Agilent Whole Human Genome Oligo Microarray.
Statistical analyses were performed by using the SPSS software. c2 test was used to analyze the correlations between SphK2 expression and the clinicopathologic features of EOC. Kaplan–Meier curves and log-rank tests were used to analyze the survival data. The values were presented as the mean ± SD and were analyzed by t-test. A P value less than 0.05 was considered significant.