From January 2015 to August 2018, ten samples of noncancerous inflammatory nasopharyngeal epithelial tissues and 109 primary NPC tumor tissues were obtained from the Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology. All tissues were collected at the first diagnosis. We divided the patients into high and low groups by taking the median score as the cut-off value. Two independent histopathologists identified the diagnosis of NPC in each patient. This study attained approval from the Research Ethics Committee of the Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology.
Immunohistochemistry (IHC) staining and scoring were performed as previously mentioned. The sections were incubated with FERMT1 antibody (22215-1-AP, 1:200 dilution, Proteintech). Regarding FERMT1 expression, these sections were then subdivided into two categories: 0 and 1+ were defined as low expression, and 2+ and 3+ were defined as high expression. In addition, tissue IHC was used to detect FERMT1, E-cadherin, N-cadherin, vimentin, CDK4, CDK6, cyclinD1 and cyclinB1 expression with the indicated antibodies for xenograft mouse tissues, including anti-FERMT1 (22215-1-AP, Proteintech, China), anti-E-cadherin (14472, Cell Signaling Technology, USA), anti-N-cadherin (22215-1-AP, Proteintech, China), anti-vimentin (22215-1-AP, Proteintech, China), anti-CDK4 (22215-1-AP, Proteintech, China), anti-CDK6 (22215-1-AP, Proteintech, China), anti-cyclinB1 (22215-1-AP, Proteintech, China), and anti-cyclinD1 (22215-1-AP, Proteintech, China).
Human NPC cell lines (CNE2, HK1, CNE1, HNE1, HONE1) and nasopharyngeal immortal epithelial cells (NP69) were acquired from the Cancer Research Institute of Central South University (Changsha, China). CNE2, HONE1 and HNE1 are epithelial cell lines of poorly differentiated nasopharyngeal squamous cell carcinoma. CNE1 is a well-differentiated squamous cell carcinoma, and HK1 is a well-differentiated, EBV-positive squamous cell carcinoma. CNE2 cell lines were identified by STR DNA profiling analysis. All NPC cell lines were routinely tested for mycoplasma contamination and cultured in RIPM 1640 medium (Promoter Biotech, China) supplemented with 10% fetal bovine serum (FBS, Gibco, South America), while NP69 cells were cultured with keratinocyte-SFM medium with 0.2 μg/ml EGF (Gibco, USA) and 30 μg/ml bovine pituitary extract (Gibco, USA). All cells were cultured in a humidified atmosphere of 37 °C and 5% CO2.
Construction of the lentivirus vectors and cell transfection
The lentiviral vector was constructed and used for cell transduction as described previously to establish stably transfected cell lines for subsequent experimentation. FERMT1 shRNA lentiviral particles were obtained from GeneChem (GV112, Shanghai, China). CNE2 and HK1 cells were transfected with FERMT1 shRNA lentiviral particles according to the manufacturer’s instructions. Puromycin (3 µg/ml, Sigma, USA) was used to select stable cell lines in selective medium for at least 2 weeks. Western blotting and real-time PCR were used to detect the efficiency of transfection. The target sequences of FERMT1 shRNA were as follows:
shRNA 1: CAGCTTCAGGTTCATCAGTAA;
shRNA 2: GAGCAGCTGCTCTTACGATTT;
shRNA 3: CAGCTCTACAGTACCACATTA.
To evaluate the protein expression, we performed western blotting. Total protein extracts were prepared with radioimmunoprecipitation assay (RIPA) buffer (P0013K, Beyotime Biotechnology, China). The proteins were measured in a microplate reader (Synergy H1, Biotek, USA) by Beyotime protein assay reagent using a wavelength of 562 nm. The proteins were separated using sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Blots were incubated with the primary antibodies at 4 °C overnight. The next day, the blots were incubated with secondary antibodies for 1 h after washing and visualized with West Dura extended duration substrate (34580, Thermo Fisher Scientific, USA). Anti-FERMT1 (22215), anti-CDK4 (11026), anti-CDK6 (14052), anti-cyclinD1 (60186), anti-N-cadherin (22018), anti-vimentin (10366), anti-cyclinB1 (55004), anti-ZEB1 (21544) and anti-GAPDH (60004) were purchased from Proteintech (Wuhan, China). Anti-E-cadherin (#14472) and anti-phospho-Rb (#9301) were obtained from Cell Signaling Technology (USA). Anti-snail (ab117866) and anti-twist1 (ab50887) antibodies were purchased from Abcam (USA). Anti-NLRP3 (M035175F), anti-total-NF-kB (T55034F), anti-Phosphorylated-NF-kB (p-NF-kB) (TP56371F), anti-total-IkBα (T55026F) and anti-Phosphorylated-IkBα (p-IkBα) (TP56280F) were obtained from Abmart (Shanghai, China).
Reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR)
According to the manufacturer’s instructions, total RNA was prepared using TRIzol reagent (Takara, Dalian, China). cDNA synthesis was carried out using HiScript II Q RT SuperMix for qPCR (Vazyme Biotech, Nanjing, China). Real-time PCR was performed with a fast real-time PCR system (7900HT, Applied Biosystems, USA). qPCR was used to detect the mRNA expression level using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China). The sequences of the primers were shown in Additional file 1: Table S1.
Cell counting kit‐8 (CCK8) assay
First, NPC cells were implanted at 800 cells per well into 96-well plates. For the CCK-8 assay (MCE, HY-K0301), CCK-8 reagent (10% of the serum-free medium volume) was added to the cells and incubated for another 1 h at 37 °C. The absorbance was determined by a microplate reader (BioTek, Winooski, VT, USA) at 450 nm to assess the cell proliferation ability. Cell viability was measured daily for six consecutive days.
Colony formation assay
The growth of NPC cells was evaluated with a colony formation assay. NPC cells were uniformly implanted at 200 per well in 6-well plates and incubated at 37 ℃ for 10–14 days. More than 50 cells under the microscope were regarded as valid clones. Then, the colonies were fixed with methanol for 20 min and stained with 0.1% crystal violet for 20 min. Three replicates were set for each group. The colonies were photographed and counted by three researchers independently. GraphPad Prism 8 software was used to analyze all data.
The migration ability of NPC cells was detected with a wound-healing experiment. The cells were plated in 24-well culture plates with RPMI 1640 medium supplemented with 10% FBS and were cultured at 37 °C until cell monolayer confluence. A straight line was drawn at the bottom of the 24-well plate with a 20-µL pipette tip, and then the cells were grown at 37 °C in serum-free RPMI 1640 medium. The migration of the cells was captured under a microscope at 0, 12, and 24 h after scratching. The scratch distances were determined by ImageJ analysis software (National Institutes of Health, USA). Each experiment was performed at least three times.
Transwell migration and invasion assay
NPC cell migration and invasion were determined by Transwell migration and invasion assays. Cell suspensions with 200 µL serum-free RPMI 1640 medium were implanted 8-μm-pore with or without Matrigel-coated Transwell chambers (Corning Costar, Cambridge, MA) to a density of 1 × 105 cells/well, and then the inserts were held in the lower chamber with 500 μL of RPMI 1640 medium supplemented with 10% FBS and incubated at 37 °C. After 36 h, cells that were on the inside of the Transwell inserts were wiped with a cotton swab. Then, cells that had migrated to the lower surface of the membrane were fixed and stained with crystal violet. Five random fields were photographed, and the cells were counted to calculate the average number of migrated cells. Each experiment was performed at least three times.
Cell cycle analysis
The cell cycle distribution was assessed by the cell cycle detection kit (Promoter, Wuhan, China) of NPC cells after silencing FERMT1. Cells were digested, collected, and fixed overnight using ice-cold 75% ethanol at − 20 °C. According to the manufacturer’s instructions, the cells were treated with RNaseA at 37 °C for 30 min and stained with propidium iodide (PI) at 37 °C in the dark for 30 min. The samples were detected by a FACSCanto II flow cytometer (BD Biosciences, USA).
Co-immunoprecipitation (co-IP) assays were performed as previously described. NPC cells were harvested and lysed with cold co-immunoprecipitation buffer (Promoter, Wuhan, China). The supernatants were subjected to immunoprecipitation with anti-FERMT1 antibody or anti-NLRP3 antibody conjugated with protein A/G magnetic beads (MCE, HY-K0202). Bound protein was detected by the western blotting analysis mentioned above.
In vivo xenograft assay
The results were further validated using a xenograft mouse model. Four- to five-week-old female BALB/c nude mice (GemPharmatech, Nanjing, China) were reared in specific pathogen-free (SPF) conditions, constant temperature and constant humidity, and sufficient food and water every day. According to the Animal Study Guidelines of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology Animal Care Facility and National Institutes of Health guidelines, all animal experiments were strictly raised. The mice were randomized into two groups, and each group contained eight mice: named HK1 and HK1-shFERMT1 groups. NPC cells (2.0 × 106/100 µL) were implanted subcutaneously into the right axilla of each mouse. The size of the tumor was measured using a caliper every 3 days. The tumor volume calculation formula was as follows: tumor volume = (long length × short length2)/2. The experiment was terminated after 27 days of tumor growth. The anesthetized mice were euthanized by cervical dislocation. This study was reviewed and approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.
The collection and analysis of data
We downloaded the expression profile of genes from the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/), and the download data format was MINIML. GSE12452 was composed of mRNA of laser-captured epithelium from 31 NPC and 10 non-NPC nasopharynx tissues from the Taiwanese case–control cohort. mRNA expression was analyzed with Affymetrix Human Genome U133 Plus 2.0 Array. Quantile normalization of microarray data was used for the log2-transformed intensity values as a method for between array normalization to obtain a similar intensity distribution across arrays. The Cancer Genome Atlas (TCGA) databases (https://www.cancer.gov/tcga/) was used and downloaded to analyze the FERMT1 in the head and neck squamous cell carcinoma (HNSCC).
Gene set enrichment analysis
According to the expression of FERMT1, gene set enrichment analysis (GSEA) was performed to identify the significantly different genes. GSEA software (UC San Diego and Broad Institute, San Diego, CA, USA) was used to analyze the GSEA of hallmarks. Gene set permutations were used 1000 times per analysis. The normalized enrichment score (NES), nominal P value, and false discovery rate (FDR) q-value indicated the significance of the enrichment results.
RNA sequencing analysis
After lentivirus FERMT1-shRNA was transfected into HK1 cells, the total RNA of each sample was extracted. The RNA concentration and purity were determined with a NanoDrop2000 (Thermo Fisher, USA). RNA sequencing (RNA-Seq) analysis was conducted by GeneChem (Shanghai, China). Differential expression gene (DEG) analysis was performed using DESeq2 and EdgeR software. Finally, padj value < 0.05 and |log2foldchange|> 2 were regarded as the thresholds for defining differential expression.
SPSS software (version 26.0; SPSS, Chicago, IL, USA) was used for statistical analysis. Means ± standard deviation (SD) were used to express the data. The chi-square test or Fisher’s exact test was applied to analyze the relationship between FERMT1 expression and clinicopathological characteristics. Kaplan–Meier analysis and a log-rank test were used for survival comparison. Statistical significance was determined with Student’s t test or two-way analysis of variance (ANOVA). Differences were considered statistically significant at a P value < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001).