The human tongue squamous cell carcinoma cell lines, CAL27 and SCC25, were kindly provided by Professor Liang Jiang (Tongji Hospital, Hubei, China). The CAL27 and SCC25 cell lines were cultured in complete DMEM (Hyclone; Cytiva) containing 10% FBS (Lonza Group, Ltd.) with 100 U/ml streptomycin and penicillin (Thermo Fisher Scientific, Inc.). Both the cell lines were cultured at 37°C in a humidified incubator with 5% CO₂. The CAL27 and SCC25 cell lines were harvested using 0.25% trypsin-EDTA (Thermo Fisher Scientific, Inc.) when it reached 80% confluence.

The CAL27 and SCC25 cell lines were transfected with small interfering (si)RNA targeting COPS5 (5′-UUCUCAUACUGUCUUUCAGGUCUGAAAGACAGUAUGAGAAAA-3′) or COPS6 (5′-UUGAUUAUAUCAAUGACAGACCUGUCAUUGAUAUAAUCAAUG-3′) or a non-specific control (50 nM) using Lipofectamine^® 2000 transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After cell transfection with siRNA for 4 h at 37°C, the medium was replaced. After 48 h transfection, cells were used for western blot analysis and wound healing assay. After 24 or 48 h transfected, cell viability was tested.

Proteins from CAL27 and SCC25 cells were extracted using RIPA lysate (Beyotime Biotechnology, China) containing 1× protease inhibitor (Thermo Fisher Scientific, USA) at 80–90% density. The protein concentration was determined by a BCA method (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Total protein (20 µg) was separated using 10% SDS-PAGE and transferred to 0.2-µm PVDF membranes (Bio-Rad Laboratories) at 100 V for 80 min. After being blocked with TBS containing 0.1% Tween-20 (TBST) and 5% skimmed milk for 1 h at room temperature, the PVDF membrane was incubated with the primary antibody overnight at 4°C. The next day, after being washed 3 times with TBST, the membrane was incubated with the goat anti-mouse secondary antibody (cat. no. SA00001-1; 1:10,000; ProteinTech Group, Inc.) for 1 h at room temperature, then the bands were visualized using an ECL developing system (Beyotime Institute of Biotechnology). The following antibodies were used: Mouse monoclonal anti-COPS5 (cat. no. sc-13157; 1:200; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-COPS6 (cat. no. sc-137153; 1:200; Santa Cruz biotechnology, Inc.) and mouse monoclonal anti-GAPDH (cat no. ab8245; 1:10,000; Abcam).

Following transfection with the siRNA, the cell lines (1×10⁴) were collected and seeded in 96-well plates. The cells were then incubated with complete medium for 24 or 48 h and 10 µg Cell Counting Kit-8 reagent (MedChemExpress) was added into each well. The cells were incubated in 37°C for 30 min, then the samples were measured at 450 nm.

The CAL27 and SCC25 (1×10⁵) cell lines were seeded in 12-well plate. Then, cells were transfected with siRNA or control. After the cells were cultured to 80–90% confluence with complete DMEM medium with 10% FBS, the cells were scratched slightly, then washed with PBS three times. Subsequently, the cells were incubated with DMEM without FBS for 48 h and images of the cells were captured with normal light microscope (BX51; Olympus Corporation). The wound was quantified by ImageJ with the Wound Healing Coherency Tool (version 1.8.0; national Institute of Health).

The RNA expression profile and clinical data sets were obtained from The Cancer Genome Atlas (TCGA) database (https://gdc.cancer.gov), which included 520 patients with HNSCC and 44 adjacent normal samples. After excluding the samples with survival times <30 days, missing clinical information and data integration, a total of 490 samples were used to perform the prognostic analysis.

The ONCOMINE database (www.oncomine.org) is the world's largest oncogene chip database and integrated data mining platform. A total of 11 datasets (Pyeon Multi-cancer, Sengupta Head-Neck, Toruner Head-Neck, Schlingemann Head-Neck, Cromer Head-Neck, Ginos Head-Neck, Giordano Thyroid, Peng Head-Neck, Ye Head-Neck, Vasko Thyroid and He Thyroid) were included in the analysis (30–35). The mRNA expression level of the COPS subunits in the clinical HNSCC specimens were compared with that in adjacent normal controls, using an unpaired Student's t-test. The fold change and P-value thresholds were defined as 1.5 and 0.01, respectively. TCGA gene expression data of the COPS subunits was determined using UALCAN (http://ualcan.path.uab.edu) using the ‘TCGA-HNSC’ module, which provides systematical and personalized analysis of TCGA database. University of California Santa Cruz (UCSC) Xena (http://xenabrowser.net/) is a comprehensive web analysis tool which provides customized analysis methods of TCGA data. To evaluate different mRNA expression levels of the COPS subunits and the TMN stages in primary HNSCC (n=765), UCSC Xena was used with a one-way ANOVA analysis followed by Bonferroni's post hoc test.

The Kaplan-Meier plotter (www.kmplot.com) includes the 21 types of TCGA datasets to evaluate the prognostic value of 54,000 genes in different types of cancer. The association between mRNA expression levels of all the COPS subunits and prognosis in patients with HNSCC was analyzed using the Kaplan-Meier plotter. All the patients were divided into two groups automatically using the best outcome setting. The hazard ratio (HR) with 95% confidence intervals (CI) and log-rank were obtained to evaluate the prognostic difference in patients with HNSCC.

To construct the PPI network of the COPS subunits, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; http://string-db.org/), which includes both certified and predicated links, was used. In total, 10 COPS subunits (COPS1-6, 7A and B, 8 and 9) and Homo sapiens (organism) were selected for the analysis. Extra 50 associated nodes were included in the analysis.

GO enrichment analysis was performed using The Database for Annotation, Visualization and Integrated Discovery (DAVID; http://david.ncifcrf.gov/). The catalogues of GO analysis were selected as biological process (BP), cellular components (CC), molecular function (MF) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was also performed. All associated genes (36) were included in the analysis. P<0.05 was considered as significant. The top 12 affected pathways were included using R version 4.1.0. (R Development Core Team).

To evaluate the importance of the COPS subunits in survival of the HNSCC cell lines, Project Achilles with CERES dependence scores was used. By accurately removing the expression of the corresponding gene with CRISPR-Cas9 system, Project Achilles systematically investigates and identifies the numbers of genes which are essential for survival of the cancer cell lines. The original data was downloaded from the Depmap portal (https://depmap.org/portal) (37). The inclusive criteria were as follow: i) The primary disease was head and neck cancer and ii) the pathological subtype was squamous cell carcinoma. A total of 21 cell lines were included in the analysis.

GSEA (http://www.broad.mit.edu/gsea), with the annotated Hallmark effector gene sets, was performed according to the mRNA expression level of the COPS subunits in TCGA-HNSC dataset. The cut-off value of grouping was defined as 50%. The false discovery rate q-value <0.25 and P<0.05 was considered to indicate a significant difference (38). The visual results were performed using R version 4.1.0.

The protein expression of COPS subunits was evaluated using immunohistochemistry (IHC) data downloaded from the Human Protein Atlas (HPA) database (https://www.proteinatlas.org/). The following primary antibodies against CCT5 and CCT6 were used CAB004242 and HPA044315.

The HR with log-rank test was used for survival analysis. Spearman's rank correlation test was used to evaluate the correlation of gene expression in tumor tissues compared with normal tissues, and the strength of the association was determined using the absolute values. One-way ANOVA followed by Bonferroni post hoc test was used to evaluate the difference in >2 groups. P<0.05 was considered to indicate a statistically significant difference.

A total 10 COPS subunits (COPS1-9) have been identified in human cells (39,40). To evaluate the mRNA expression levels of the COPS subunits in HNSCC, TCGA dataset (TCGA-HNSC) was used within the UALCAN database. As shown in Fig. 1, the expression of COPS1-8, but not COPS9, was found to be upregulated in HNSCC tissues compared with that in normal tissues.

To further verify the results, the ONCOMINE database was used to compare the transcriptional levels of the COPS subunits in all types of cancer with the corresponding normal tissue (Fig. 2). The red color and number indicated the number of datasets with a statistically significant increase (P<0.01; fold change, 1.5) in mRNA expression levels in different types of cancers, while the blue color indicated a decrease in mRNA expression of the COPS subunits. There was a total of 11 HNSCC datasets in the ONCOMINE database. The overexpression status of COPS subunits in different parts of HNSCC are presented in Table I. In detail, one dataset indicated COPS1 was upregulated in floor of the mouth carcinoma, and COPS3 was increased in carcinoma from oropharyngeal, oral cavity, floor of the mouth and tongue (Pyeon Multi-cancer) (30). The expression of COPS2 was found increased in both NPC (Sengupta Head-Neck) (31) and floor of the mouth carcinoma (Pyeon Multi-cancer) (30). COPS5 (Pyeon Multi-cancer, Sengupta Head-Neck and Toruner Head-Neck Statistics) (30,31,34) and COPS6 (Pyeon Multi-cancer) (30) were upregulated in various sites of HNSCC. In addition, COPS8 was found highly expressed in HNSCC tissues in five datasets (Pyeon Multi-cancer, Sengupta Head-Neck, Schlingemann Head-Neck, Cromer Head-Neck and Ginos Head-Neck) (30–33,35). There was no significant difference in the expression of COPS7A, COPS7B and COPS9 between HNSCC and normal tissues in the ONCOMINE database.

As most of the COPS subunits were found to be upregulated in HNSCC tissues, the association between the mRNA expression level of the COPS subunits and TNM stage was investigated. The UCSC Xena web tool was used for the analysis. As shown in Fig. 3A, the mRNA expression levels of COPS5 and COPS6 were significantly increased with increasing tumor size. With respect to lymph node metastasis, an increase in the expression level of COPS5, COPS6, COPS7B and COPS9 were found in patients with HNSCC and N stage (Fig. 3B). In addition, increased expression of COPS5, COPS6, COPS8 and COPS9 was associated with distant metastasis of HNSCC (Fig. 3C).

The Kaplan-Meier plotter database and TCGA-HNSC dataset was used to investigate whether the COPS subunits were associated with the prognosis of HNSCC. High mRNA expression level of COPS2 (HR, 1.44; CI, 1.09–1.9; P=0.0091; Fig. 4B), COPS5 (HR, 1.32; CI, 1.01–1.73; P=0.042; Fig. 4E), COPS6 (HR, 1.4; CI, 1.06–1.84; P=0.019; Fig. 4F), COPS7A (HR, 1.56 (1.19–2.05); P=0.0011; Fig. 4G), COPS8 (HR, 1.6; CI, 1.16–2.19; P=0.0035; Fig. 4I) and COPS9 (HR, 1.33; CI, 1.02–1.74; P=0.035; Fig. 4J) was associated with poor prognosis in patients with HNSCC. However, high expression of COPS7B (HR, 0.75 (0.57–0.98); P=0.038; Fig. 4H) was associated with improved outcome in patients with HNSCC.

To predict the functions of the COPS subunits in humans, a PPI network was constructed with the 50 most relevant genes using STRING. The analysis included 60 nodes and 946 edges (Fig. 5). The PPI enrichment value was P<1.0×10⁻¹⁶.

Then, the biological functions and pathways of the associated genes with the COPS subunits were analyzed using GO and KEGG analysis, respectively. The top 12 GO terms and KEGG pathways are shown in Fig. 6. With respect to BP, ‘protein ubiquitination involved in ubiquitin-dependent protein catabolic process’, ‘Fc-epsilon receptor signaling pathway’, ‘nucleotide-excision repair, preincision complex stabilization’ and ‘G₁/S transition of mitotic cell cycle’ were significantly associated with the COPS subunits (Fig. 6A; Table SI). For CC, the genes were associated with ‘Cul3-RING ubiquitin ligase complex’, ‘nucleoplasm’ and ‘cytosol’ (Fig. 6B; Table SII). The genes associated with MF were ‘ubiquitin-protein transferase activity’, ‘MAP kinase activity’, ‘NEDD8 transferase activity’ and ‘JUN kinase activity’, which were cancer relative pathways (Fig. 6C; Table SIII). KEGG pathway analysis identifies the functions of the COPS subunits and the pathways they are involved in. The top 12 pathways are shown in Fig. 6D and Table SIV. Among these pathways, ‘ubiquitin mediated proteolysis’, ‘renal cell carcinoma’, ‘pathways in cancer’, ‘colorectal cancer’, ‘Toll-like receptor signaling pathway’ and ‘TNF signaling pathway’ were observed.

In HNSCC, COPS5, COPS6 and COPS8 are essential for cell growth and, as aforementioned, associated with tumor TNM stages and prognosis. Hence, it is important to evaluate the functions of these three genes and GSEA was performed to evaluate Hallmark annotation. As shown in Fig. 7A and Table SV, high expression of COPS5 was associated with tumorigenesis-associated pathway including ‘DNA repair’, ‘unfolded protein response’, ‘MTORC1 signaling’, ‘glycolysis’ and ‘PI3K AKT mTOR signaling’. In addition to these pathways, increased COPS6 was also associated with ‘oxidative phosphorylation’ and ‘p53 pathway’ (Fig. 7B and Table SVI). Similarly, COPS8 was additionally associated with ‘apoptosis’ (Fig. 7C and Table SVII).

The protein expression level of the COPS subunits in HNSCC was investigated using the HPA database. Consistent with the mRNA expression level, COPS2 (Fig. S1A), COPS3 (Fig. S1B), COPS4 (Fig. S1C), COPS5 (Fig. 8A), COPS6 (Fig. 8B), COPS7A (Fig. S1D) and COPS7B (Fig. 1E) were notably upregulated in both the cytoplasm and the nucleus in the HNSCC tissues compared with that in normal oral epithelial tissues. COPS9 was increased in the normal oral epithelial tissue compared with that in the tumor tissues of HNSCC (Fig. S1F). COPS1 and COPS8 were not detected.

To evaluate the importance of the COPS subunits, Project Achilles with CERES dependence scores was used. When the score was >-1, it indicated that the gene was not essential for cell survival. Otherwise, the gene was considered important for cell survival. As shown in Fig. 9A and Table SVIII among all the HNSCC cell lines, COPS6 was important for cell survival. COPS1, COPS3, COPS4, COPS5 and COPS8 were critical for cell survival in most HNSCC cell lines. COPS2 was significantly essential for only 2 HNSCC cell lines. COPS7A, COPS7B and COPS9 were not important for HNSCC cell survival. These results suggested that COPS1, COPS3, COPS4, COPS5, COPS6 and COPS8 are essential for HNSCC cell survival.

Taken together, COPS5 and COPS6 were associated with the survival of the HNSCC cells and could be detected in tumor samples using IHC. To confirm the biological function of COPS5 and COPS6 in HNSCC, the CAL27 and SCC25 cell lines were transfected with a control siRNA, si-COPS5 and si-COPS6. Western blot analysis showed that the protein expression level of COPS5 (Fig. 9B and D) and COPS6 (Fig. 9C and E) was notably decreased by the siRNA. As shown in Fig. 9F and G, the cell viabilities of the CAL27 and SCC25 cell lines were significantly decreased by both si-COPS5 and si-COPS6 at 24 and 48 h time points (P<0.05). To detect the cell migration effect, the CAL27 and SCC25 cell lines were transfected with control siRNA, si-COPS5 or si-COPS6. The results from the wound healing assay suggested that si-COPS5 and si-COPS6 inhibited the migration of the CAL27 and SCC25 cells (Fig. 9H and I). These results suggested that COPS5 and COPS6 could be essential for growth and migration of the HNSCC cell lines.