Patients and samples
Twenty-five patients with OSCC underwent surgical resection at the Department of Craniofacial Surgical Resection, Stomatological Hospital, Southern Medical University, Guangzhou, China. Primary OSCC tissues (n = 25) and some adjacent normal tissues (n = 10) were obtained postoperatively. All patients provided written informed consent prior to enrolment in the study. The study protocol was approved by the Ethics Committee of Stomatological Hospital, Southern Medical University. Another 18 OSCC tissue samples were acquired from tissue chips with detailed clinical information and were purchased from WoZhe Biotechnology Company Ltd. (Guangzhou, China).
Cell lines and reagents
The HSC6 cell line was purchased from CinoAsia Co., Ltd. (Shanghai, China). CAL27, SCC9 and HOK cell lines were purchased from TongPai Biotechnology Co., Ltd. (Shanghai, China). OSCC cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin, and HOK cells were cultured in KSFM (Gibco, USA). Cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C. Recombinant human CCL18 (rCCL18) was obtained from Peprotech (Princeton, NJ, USA). The JAK2/STAT3 signaling pathway specific inhibitor AG490 was purchased from Selleck Chemicals (Houston, TX, USA).
The OSCC tissues and adjacent normal tissues each were analyzed using immunohistochemistry (IHC). In brief, tissues were dewaxed in xylene and rehydrated using a graded alcohol series. After antigen retrieval with Tris-EDTA, the slides were blocked with 5% serum. Primary antibodies against CCL18 (1:100, Santa Cruz Biotechnology Inc., USA), NIR1 (1:100, Novus, Littleton, CO, USA), CCR6 (1:100, Novus, Littleton, CO, USA) and CCR8 (1:100, Abcam, UK) were incubated overnight at 4 °C. Then, the sections were covered with secondary antibody and incubated at room temperature for 30 min. Next, the tissue sections were visualized with DAB (Gene, Shanghai, China). The staining results were evaluated using a visual grading system based on the average optical density scored using the following criteria: the percent score of positive cells: 0 (< 5%); 1 (5–25%); 2 (26–50%); 3 (51–75%); 4 (76–100%); the staining intensity: 0 (negative), 1 (weak), 2 (moderate), 3 (strong). Positive grade = percentage score × staining intensity score. Specifically, 0–1 was considered as (−), 2–8 as (+), 9–12 as (++).
Cells were seeded in glass bottom cell culture dishes for 24 h. Thereater, the cells were rinsed with PBS, fixed with 4% paraformaldehyde solution for 30 min, permeabilized with 0.3% Triton X-100 for 15 min, and blocked with 5% bovine serum albumin (BSA) for 1 h. Subsequently, the cells were incubated overnight at 4 °C with the following primary antibodies: NIR1 (1:200, Novus, USA), CCR6 (1:200, Novus, USA), and CCR8 (1:100, Abcam, UK). The next day, the samples were incubated with secondary antibody (1:500, Abcam, UK) in the dark for 1 h and counterstained with DAPI (Invitrogen, USA) for 5 min. The results were photographed using an automated upright microscope system (Leica, DM4000B Leica Microsystems, Wetzlar, Germany).
Transfection of NIR1 siRNA
For transfection, HSC6 cells and CAL27 cells were seeded in 6-well plates at 2 × 105/well. siRNA against NIR1 (siNIR1) was transferred into cells with Lipofectamine 2000 (Invitrogen, USA), used according to the manufacturer’s instructions. A negative siRNA (siNC) sequence was used as a control. Silencing efficiency was verified by qRT-PCR and Western blot assays after 48 h of transfection. The following three interfering sequences for NIR1 were synthesized (GenePharma, Jiangsu, China):
siNIR1–1: sense 5′-CCAUCUGCUCUGAGGCUUUTT-3′ antisense 5′-AAAGCCUCAGAGCAGAUGGTT-3′.
siNIR1–2: sense 5′-CACGCCCAAAGAAGAACAATT-3′ antisense 5′-UUGUUCUUCUUUGGGCGUGTT-3′.
siNIR1–3: sense 5′-GUGGUCGCAUCACAUACAATT-3′ antisense 5′-UUGUAUGUGAUGCGACCACTT-3′.
Negative control: sense 5′-UUCUCCGAACGUGUCACGUTT-3′ antisense 5′-ACGUGACACGUUCGGAGAATT-3′.
Western blot analysis
Cells and tissues were lysed in cell lysis buffer with phosphatase inhibitor, protease inhibitor and PMSF (KeyGEN BioTECH, Jiangsu, China). Total protein levels were measured using a BCA protein assay kit (Cwbiotech, Jiangsu, China). Twenty micrograms of protein was separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Merck KGaA, Darmstadt, Germany). The PVDF membrane was blocked with 5% BSA (Pierce, Rockford, IL, USA) for 1 h and then incubated with the following primary antibodies at 4 °C overnight: NIR1 (1:2000, Novus, USA), CCR6 (1:250, Novus, USA), CCR8 (1:2000, Abcam, UK), GAPDH (1:1000, Abcam, UK), E-cadherin (1:1000, CST, Danvers, MA, USA), N-cadherin (1:1000, CST, USA), ZEB2 (1:1000, Merck KGaA, Germany), JAK2 (1:1000, CST, USA), P-JAK2 (Tyr1007/1008) (1:1000, CST, USA), STAT3 (1:1000, Sant Cruze Biotechnology Inc., USA), P-STAT3 (Tyr705) (1:1000, CST, USA), and β-actin (1:1000, Abcam, UK). Thereafter, the PVDF membrane was incubated with secondary antibody (1:2000, Abcam, UK). Protein bands were detected by ultrasensitive chemiluminescence imaging, and Image Lab software was used to analyse the density of each band.
Cells were collected, and total RNA was extracted using TRIzol reagent (Invitrogen, USA). Complementary DNA (cDNA) was synthesized using a FastKing gDNA Dispelling RT SuperMix (TIANGEN, Beijing, China). qPCR was performed using the Talent qPCR PreMix (TIANGEN, China) on a CFX96TM Connect Real-Time System (C1000 TouchTM Thermal Cycler, BIO-RAD, Hercules, CA, USA). The thermocycling conditions were as follows: 3 min at 95 °C, followed by 40 cycles of 5 s at 95 °C and 15 s at 60 °C. The relative levels of mRNA expression were normalized to GAPDH levels as the reference gene, using the 2-∆∆Cq method.
The primers sequences used were as follows: NIR1: (Forward: GATGCCAGAGGAGAAGGGAC; Reverse: TCGCTGTCTTCGTGGATCTC), GAPDH: (Forward: CTCCTCCTGTTCGACAGTCAGC; Reverse: CCCAATACGACCAAATCCGTT).
Cells were pretreated with siRNA-NIR1for 48 h or AG490 for 24 h, and 5000 cells were then added to 96-well plates and treated with 20 ng/ml rCCL18. At 24 h, 48 h and 72 h, CCK-8 reagent (Sigma-Aldrich, Louis, MO, USA) was added, and the absorbance values of each well at 450 nm were read using a microplate reader (Thermo Fisher Scientific. Waltham, USA).
Clone formation assay
Forty-eight hours after siRNA-NIR1 transfection, cells were plated in 6-well plates at 1000 cells per well and exogenously stimulated with 20 ng/ml rCCL18 (3% FBS). The number of cell clones was counted using crystal violet staining 14 days later.
Cell migration and invasion were detected using transwell assays (Corning, New York, NY, USA). The upper chamber was precoated with 50 μl 20% Matrigel (Gibco, USA) for the invasion assay. Cells were transfected with siRNA for 48 h or treated with AG490 for 24 h. Treated cells were suspended in serum-free medium with or without 20 ng/ml rCCL18. The prepared cells were seeded in the upper insert, and the lower chamber was filled with DMEM containing 15% FBS. Then, the transwell plates were incubated at 37 °C with 5% CO2 for 24 h. Cells that did not invade through the pores were gently removed with cotton tips. The upper chamber was fixed with 4% formaldehyde for 15 min and stained with a 0.4% crystal violet solution for 15 min. Five randomly selected fields of view at × 50 magnification were photographed under a light microscope (Carl Zeiss AG, Oberkochenm, Germany) and analyzed.
Statistical analysis was performed using GraphPad Prism 7.00 software (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS version 20 (IBM Corporation, Armonk, NY, USA). The data are presented as means±SEM based on three replicates per group. Chi square tests was used to analyze the association of NIR1 with the clinical variables of OSCC patients. Student’s t test and one-way ANOVA were used to compare the mean differences between different sample group. P < 0.05 was considered statistically significant.