Human NPC specimens
We obtained the paraffin-embedded NPC specimens from Affiliated Tumor Hospital of Nantong University. All the samples were confirmed by pathological diagnosis as nasopharyngeal squamous carcinoma. Patients in group were newly diagnosed without any special treatments (including radiation and chemotherapy), excluding organ dysfunction (including liver, kidney, cardiovascular, etc.) or concurrent with other malignant tumors. Non-cancerous samples in nasopharynx were used as controls. Patients' clinical information were shown in table 1. Fresh biopsy samples were reserved under -80 °C and all specimens got patients' informed consent. The research was approved by the Ethics Committee of Affiliated Hospital of Nantong University.
IHC was implemented as previously described. Tissue sections were incubated with anti-human CD73 (Sangon Biotech, D121879, 1:50) and immunoreactivity of CD73 was visualized with 3, 39-diaminobenzidine tetrachloride (DAB) chromogenic solution. IHC scores were evaluated by two pathologists without knowing patients’ clinicopathological outcomes. The intensity of CD73 staining was scored as 0, no staining;1, weak staining;2, medium staining;3, strong staining. The percentage of immunopositive cells was scored as 0, <10%; 1,10–25%; 2,26–75%; 3, >75%. Then we summed the previous two scores and classified as: low expression group: 0-3, high expression group:4-6.
Cell lines we used included NPC cells CNE1(high differentiation), CNE2(low differentiation), 5-8F(high tumorigenesis and high metastasis), 6-10B(low tumorigenesis and low metastasis) and normal nasopharyngeal epithelial cells NP69. They were generously given by the Sun Yat-Sen University and Xiang-Ya School of Medicine. All the tumor cells were growing in RPMI 1640 (Biological Industries Israel Beit-Haemek, 01–100-1ACS) with10% fetal bovine serum (Biological Industries Israel Beit-Haemek, 04–001-1ACS), NP69 was cultured in Keratinocyte-SFM (Thermo Fisher Scientific, 17005–042). Cells were cultured at 37 °C in 5% CO2 incubator.
We took western blot to detect protein expression of CD73. Tissues and cells were lysed with RIPA Lysis Buffer and protease inhibitors. After quantified the concentration of proteins with a Pierce Bicinchoninic Acid (BCA) protein assay kit (Thermo Fisher Scientific), the proteins were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then they were transferred to polyvinylidene difluoride membranes (Millipore, ISEQ00010, Bedford, MA). Membranes were incubated in fat-free milk and incubated with anti-CD73(Sangon Biotech, D121879, 1:500) overnight. In the next day, after incubating the membrane with HRP-tagged secondary antibody, we visualized the band with ECL reagent (Millipore).
Total RNA was extracted with Trizol reagent (Thermo Fisher Scientific, 15596018) and then it was reverse transcribed into cDNA. We performed qRT-PCR with SYBR Green PCR Master Mix (Roche, 04913914001) in a Real-Time PCR System (StepOne Plus, Applied Biosystems, Grand Island, NY) with the following primers:CD73: forward: 5′- TGCCCAGTTATGACCCTCTC -3′, reverse: 5′- GGAACCCATCTCCACCATTG -3′. The results were normalized with GAPDH and relative mRNA expression were quantified by 2-ΔΔCt method.
Transfection with siRNAs
Cells were cultured in plates at an appropriate density, then they were transfected with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. CD73-siRNAs were designed and obtained from Biotechnologies Co, Ltd (Nantong, China). CD73-siR1, Sense: 5′-GGAUACACUUCCAAAGAAAdTdT-3′, Antisense: 5′-UUUCUUUGGAAGUGUAUCCdTdT-3′; CD73-siR2: Sense: 5′-CCUGAAGUAGAUAAGUUAAdTdT-3′, Antisense: 5′-UUAACUUAUCUACUUCAGGdTdT-3′; CD73-siR3: Sense: 5′-GAAGAUCGAGUUUGAUGAAdTdT-3′, Antisense: 5′-UUCAUCAAACUCGAUCUUC dTdT-3′; CD73-siR4: Sense: CGGAUGAAAUGUUCUGGAA dTdT-3′, Antisense: 5′-UUCCAGAACAUUUCAUCCG dTdT-3′.
NPC cells were transfected with CD73-siRNA or non-specific siRNA, then they were seeded into a 96-well plate (Corning inc, Corning NY) at the density of 5000 cells per well. At different time points, 10ul CCK8 solution (Beyotime Institute of Biotechnology, China) was added to each well. Optical density(OD) was accessed with a microplate reader at 450 nm.
Colony formation assay
500 cells which had transfected with si-CD73 or si-NC were seeded on 6-well plates. After incubation for 10 days, cells were fixed with 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet solution. Colonies that contain more than 50 cells were counted.
5 × 104 pretreated cells (transfected with si-CD73 or si-NC) were resuspended in 200 ul serum-free medium and seeded into the upper chamber of an 8mm polycarbonate flitter (Millipore). The lower chamber was filled with culture medium with 10% FBS. Following the incubation of 18 h, cells in the upper surface were removed and cells adhering on the lower surface were fixed in 4% paraformaldehyde. The migration cells were visualized under a microscope after staining with 0.1% crystal violet.
Wound healing assay
CNE2 or CNE1 cells transfected with si-CD73 or si-NC were seeded on 6-well plates. When cells confluence reached about 80%, the scratches were created with a 100 ul pipette tip. Then cell culture medium was replaced with serum-free medium. At different time points, the migration distance of cells was captured. Percentage of wound width was calculated by the wound width/the distance measured at 0 h.
To analyze the expression level of CD73 (NT5E) mRNA in Head and Neck squamous cell carcinoma (HNSC), we used GEPIA2(Gene Expression Profiling Interactive Analysis) database (http://gepia2.cancer-pku.cn/), which is a publicly available web server for analyzing RNA sequencing expression from TCGA.
All the experiments were repeated 3 times and data presented as mean±standard deviation (SD). Statistical analysis was performed with GraphPad Prism 6 and SPSS17.0. Two-tailed student’s t-tests were used to determine statistical significance in groups and χ2 test tests were used to evaluate the correlation of CD73 expression level with clinicopathological features. Kaplan-Meier curves was constructed for survival analysis, and log-rank test was performed. P value less than 0.05 was considered statistically significant.