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Evaluating H2BC9 as a potential diagnostic and prognostic biomarker in head and neck squamous cell carcinoma

European Journal of Medical Research volume 30, Article number: 54 (2025) Cite this article

Abstract

Background

Histone H2B is highly expressed in many types of cancers and is involved in cancer development. H2B clustered histone 9 (H2BC9), a member of the H2B family, plays critical roles in gene expression regulation, chromosome structure, DNA repair stability, and cell cycle regulation. However, the diagnostic and prognostic value of H2BC9 in head and neck squamous cell carcinoma (HNSCC) remains unclear. This study aimed to evaluate the potential diagnostic and prognostic value of H2BC9 in HNSCC and investigate its biological role using bioinformatics.

Methods

The expression pattern and diagnostic value of H2BC9 in HNSCC were explored using UCSC Xena and GEO database. H2BC9 expression was validated using the Human Protein Atlas database, qRT-PCR, and western blotting. Prognostic value was assessed using Kaplan–Meier curves, Cox regression analysis, and a nomogram. Drug sensitivity was predicted using the R package pRRophetic, and molecular interactions were analyzed using the DepMap database. The impact of H2BC9 on HNSCC cells was further investigated through in vitro experiments.

Results

H2BC9 was markedly upregulated in HNSCC cell lines and tissues. High expression of H2BC9 was correlated with advanced-stage disease and poor prognosis. KEGG analysis linked H2BC9 to cell cycle regulation and DNA replication. H2BC9 expression influenced the drug sensitivity of paclitaxel, docetaxel, cisplatin, and 5-fluorouracil. Key molecules, such as TONSL, PITX2, NOTCH1, and H2BC10, were positively correlated with H2BC9 expression. Silencing H2BC9 suppressed cell proliferation, induced G2/M cell cycle arrest, and enhanced apoptosis and DNA damage in HNSCC cells.

Conclusion

We demonstrated that H2BC9 expression may be associated with HNSCC development and prognosis. These findings may provide a potential therapeutic target for HNSCC.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is a diverse tumor arising from the mucosal epithelium of the oral cavity, pharynx, and larynx [1]. It is the sixth most prevalent tumor worldwide, with over 740,000 new cases and more than 360,000 deaths reported in 2020 [2]. HNSCC is characterized by a high incidence of cervical lymph node metastases, increased invasive and recurrent capabilities, and poor prognosis [1, 3, 4]. The lack of evident symptoms and reliable molecular markers for the early diagnosis of HNSCC leads to limited therapeutic effects [5]. Despite substantial progress in multidisciplinary treatments, such as surgery, radiotherapy, and chemotherapy, the 5-year overall survival (OS) of patients with HNSCC, especially those in advanced stages, remains poor [6]. Therefore, there is an urgent need to enhance prognosis by identifying new diagnostic biomarkers and therapeutic targets specific to HNSCC.

Histones, which include five types (H1, H2A, H2B, H3, and H4), bind to DNA to form chromatin in eukaryotes. The nucleosome, the basic repeating unit of chromatin, consists of two copies each of H2A, H2B, H3, and H4 [7]. H2B family genes significantly impact the prognosis of liver, lung, and breast cancers [8,9,10]. In gliomas, H2B plays a crucial role in the pathological process by affecting cell cycle and immune responses. Specifically, H2B clustered histone 9 (H2BC9) and H2B clustered histone 11 (H2BC11) have been identified as independent prognostic factors for glioma. Moreover, H2B mutations are particularly prevalent in certain cancer types, including HNSCC [11]. Previous studies have demonstrated a strong correlation between histone epigenetic modifications and metastasis in various cancers, including HNSCC [12]. This correlation is attributed to the alteration of signaling molecules in the epithelial-mesenchymal transformation pathway, regulated by histone post-translational modifications [13]. However, the functional role and biological mechanism of H2BC9 in HNSCC remain unclear.

In this study, we analyzed the expression signatures, diagnostic and prognostic value, potential molecular functions, and associated pathways of H2BC9 using multiple databases. To verify the expression of H2BC9, we used immunohistochemical results from the Human Protein Atlas (HPA) database, and conducted quantitative real-time PCR (qRT-PCR) and western blotting for mRNA and protein expression, respectively. Additionally, we investigated the drug sensitivity related to H2BC9 expression and analyzed molecules associated with H2BC9 at the cellular level. Finally, we silenced H2BC9 expression using RNA interference to explore its molecular functions in HNSCC cell lines. The results of this study contribute to a better understanding of the role of H2BC9 in cancer occurrence, development, and prognosis, particularly in HNSCC.

Materials and methods

Database and data acquisition

The Cancer Genome Atlas (TCGA) (https://genomecancer.ucsc.edu/) provides genomic, transcriptomic, and proteomic data on 33 types of cancer for >20,000 patients. Gene expression transcriptome data and clinical data were downloaded from the UCSC Xena database (https://xenabrowser.net/datapages/). The GSE30784 (including 167 oral squamous cell carcinoma tissues and 45 normal tissues) and GSE51985 datasets (including 10 laryngeal squamous cell carcinoma and 10 adjacent non-neoplastic tissue samples) were obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). Cancer drug sensitivity genomics data were downloaded from the Genomics of Drug Sensitivity in Cancer (GDSC) database (https://www.cancerrxgene.org/). Head and neck cancer cell RNAseq data were retrieved from the DepMap database (https://depmap.org/portal/download/all/). For chip expression profile data, probes that mapped to multiple genes were removed, for multiple probes corresponding to one gene, the maximum value was used for expression. TCGA-HNSCC expression profiles were processed using the transcripts per million (TPM) method and transformed using log2 (TPM + 1) for normalization. Immunohistochemical staining data for normal and oral squamous cell carcinoma tissues were downloaded from the HPA database (https://www.proteinatlas.org). As these databases are open and accessible, approval from the local ethics committee was not required.

Analysis of H2BC9 expression

R version 4.2.1 was used to compare the expression of the H2BC9 gene in tumor and adjacent normal tissues, with the “ggplot2” package adopted for visualization. The GSE30784 and GSE51985 datasets were used to verify H2BC9 expression by means of a t test.

Survival analysis

In TCGA-HNSCC tumor tissues, samples were divided into high- (n = 252) and low- (n = 252) H2BC9 expression groups based on the median expression level of H2BC9. Survival probabilities were calculated using Kaplan–Meier analysis and the log-rank test. The prognostic value of H2BC9 was further explored using the Kaplan–Meier plotter database (http://kmplot.com/analysis). Cox regression analysis was conducted to investigate the influence of different variables, including age, gender, grade, clinical stage, lymphnode neck dissection, primary therapy outcome, and H2BC9 expression, on the prognosis of patients with HNSCC. Factors with P < 0.05 based on univariate Cox analysis were included into the stepwise multivariate analysis. In addition, we established a nomogram based on prognostic factors to predict the OS of HNSCC. The “survival” R package was utilized for statistical analysis. The results were manifested as forest plots using the R software (Version 4.2.1) and “ggplot2” package, which displayed P values, hazard ratios (HRs), and their respective 95% confidence intervals (CIs). The predictive accuracy of this model was evaluated using a calibration curve. Clinical HNSCC samples from the First Affiliated Hospital of Guangxi Medical University (76 with high H2BC9 expression vs. 76 with low H2BC9 expression) were used to verify the prognostic value of H2BC9 through Kaplan–Meier curve analysis.

Enrichment and gene set enrichment analysis (GSEA) analyses

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the top 50 differentially expressed genes (DEGs), sorted by P < 0.05, using the “clusterProfiler 4.2.0” package [14]. GSEA of the Hallmark and KEGG gene sets was performed using the fgsea package (version 1.30.0). The enrichment strength was indicated using the normalized enrichment score (NES). The level of significance was defined at P < 0.05.

Drug sensitivity analysis

Sensitivity scores were used to evaluate the therapeutic response of patients with high- and low-H2BC9 expression to candidate drugs. We utilized the R software package (pRRophetic, version 0.5) to predict the sensitivity of HNSCC samples to chemotherapy. GDSC training set was used to conduct 10-fold cross-validation tests and estimate the accuracy of IC50. By applying a filter condition of P < 0.05, we determined the chemotherapy sensitivity for each tumor sample.

Molecular correlation analysis at the cell line level

Data for head and neck cancer cell lines were downloaded from the DepMap database, which integrates the Cancer Cell Line Encyclopedia and Profiling Relative Inhibition Simultaneously in the Mixtures platform [15]. Data were analyzed using k-means clustering analysis or the “Data Explorer” feature in “Tools.”

Patient samples

In total, 152 pairs of HNSCC and corresponding adjacent tissues were collected from The First Affiliated Hospital of Guangxi Medical University. The recruitment phase for the study spanned from April 1, 2013 to December 31, 2018. The inclusion criteria were as follows: (1) 18–70 years old, histopathological diagnosis of HNSCC; (2) Kanofsky performance score (KPS) ≥ 90; (3) underwent radical treatment according to the guidelines and (4) availability of regular follow-up information. The exclusion criteria encompassed: (1) history of any other cancer; (2) diagnosis with distant metastasis before treatment; (3) any prior treatment for HNSCC and (4) cognitive dysfunction. The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University and the approval number was 2024-E533-01. This study has been officially sanctioned by the Medical Ethics Committee of the First Affiliated Hospital of Guangxi Medical University, thereby waiving the requirement for informed consent from participants. The study did not involve minors. All experiments were performed in accordance with relevant guidelines and regulations. The study was conducted according to the Declaration of Helsinki.

Cell line and culture

The normal human nasopharyngeal epithelial (NP69), nasopharyngeal carcinoma (C666-1, 5-8F), normal keratinocyte (HaCaT), human larynx carcinoma (TU212 and Hep2), normal oral keratinocyte (NOK), and human oral squamous cell carcinoma (CAL-27) cell lines were obtained from the Nasopharyngeal Cancer Research Laboratory at Guangxi Medical University. NP69 cells were cultured in keratinocyte-SFM medium (Invitrogen, Carlsbad, CA, USA) supplemented with bovine pituitary extract (BD Biosciences, San Diego, CA, USA). Human HNSCC cells were cultured in RPMI-1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% streptomycin (100 mg/mL)/penicillin (100 IU/mL). All cells were incubated with 5% CO2 at 37 °C in a humidified incubator. H2BC9-small interfering RNAs (siRNAs, Hanbio, WuHan, China, 1 × 108 TU/mL), and the corresponding negative controls (Hanbio, 1 × 108 TU/mL) were transfected into Cal-27 and Hep2 cells using lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The target sequences were 5′-GTTTACAAGGTGCTGAAGCAA-3′ (siH2BC9 # 1) and 5′-TGGCTCATTACAACAAGCGTT-3′ (siH2BC9 # 2).

qRT-PCR

Total RNA was extracted from HNSCC cells using Trizol reagent (Takara Bio, Kusatsu, Japan). The mRNA was quantitatively detected using Nano Drop (Thermo Fisher Scientific, MA, USA). cDNA was synthesized using 5× MonScriptTM RTIII All-in-One Mix and dsDNase reverse transcriptase (Monad Bio, Suzhou, China). The cDNA was used for the next qPCR reaction with StepOne PlusTM RT-qPCR System (Applied Biosystems, Sheffield Hallam University, UK). Amplification cycles consisted of pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 10 s, annealing at 55–65 °C for 10 s, and extension at 72 °C for 30 s. The cycles were carried out 40 times. The relative RNA expression was determined using the 2−ΔΔCt method, with GAPDH serving as the internal control. The primer sequences used were as follows: H2BC9: forward: 5′-CGTTCGACCATCACCTCCAG-3′, reverse: 5′AGCCTTTGGGTTTGAACATGC-3′; and GAPDH: forward: 5′-AATCAAGTGGGGCGATGCTG-3′; reverse: 5′-GCAAATGAGCCCCAGCCTTC-3′.

Western blotting

Total protein was extracted from cells using the RIPA reagent, protein concentration was calculated using a BCA protein assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). The sample was mixed with 5× loading buffer, at a final concentration of 1×. All samples were denatured at 100 °C for 10 min. Protein samples (50 µg/well) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA), which were blocked with 5% nonfat milk for 2 h at 37 °C. The membranes were incubated overnight at 4 °C with a primary antibody solution containing anti-H2BC9 antibody (MA5-14835; Thermo Fisher Scientific, 1:1000) and then with a goat anti-mouse IgG(H + L) secondary antibody HRP (31430, Thermo Fisher Scientific; 1:5000) for 1 h at room temperature. Chemiluminescence detection reagents (Beyotime Biotechnology) were prepared in the dark to detect the blots.

Cell proliferation assay

Cell proliferation was assessed using the CCK-8 assay (GLPBIO). Briefly, 1 × 103 cells/well were seeded into a 96-well plate and cultured for 0, 24, 48, 72, and 96 h. CCK-8 solution (10 µL) was added to each well and incubated at 37 °C for 2 h. The optical density was measured at 450 nm using a microplate reader (Thermo Fisher Scientific). Assays were conducted with three replicates according to the manufacturer’s instructions.

Apoptosis assay

Cells were plated in six-well plates at a density of 1.5 × 105 cells/well, followed by 48 h of incubation. Then, 5 × 105 cells were harvested, resuspended in 500 μL 1× binding buffer, and stained with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) using an Annexin V-FITC/PI staining kit (Keygen, Nanjing, China). Then, the cells were incubated for 15 min at room temperature in the dark. The apoptotic cells were detected using a CytoFLEX system (Beckman Coulter, Brea, CA, USA).

Cell cycle assay

Cell cycle detection was performed according to the kit’s protocol (Keygen, Nanjing, China). Cal-27 and Hep2 cells were cultured in a six-well serum-free culture with 1 × 105 cells for 48 h. Then, cells were collected and adjusted to a concentration of 1 × 106/mL. After being fixed with 70% pre-cooled ethanol at 4 °C for 3 h, the cells were stained with 500 μL PI/RNaseA working solution (PI: RNase A = 9:1) at room temperature in the dark for 60 min. The number of cells in the G1, S, and G2 phases was counted using the CytoFLEXmsystem (Beckman Coulter).

Comet assay

A comet assay was performed using the DNA Damage Detection Kit (Keygen) following the manufacturer’s instructions. The cells were inoculated in a six-well plate. when the density reached 80–90%, cells were collected and diluted to 1 × 106 cells/mL. Then, cells were lysed in a specific lysis buffer (100 mM EDTA, 2.5 M sodium chloride, 10 mM Trizma base, and 1% N-lauroylsarcosinate, 1% Triton X-100) at 4 °C for 2 h. Next, the DNA was uncoiled and unwound in an alkalescent electrophoresis buffer (1 mmol/L EDTA and 300 mmol/L NaOH) for 30 min and then used for electrophoresis. After washing the slides with 0.4 M Tris buffer, the cells were stained with a 4′,6-diamidino-2-phenylindole solution for 10 min in a dark room. An Eclipse fluorescence microscope (Nikon, Tokyo, Japan) was used for the analysis. Tail moments were used to evaluate the degree of DNA damage using OpenComet v1.3.1 [16].

Statistical analysis

The expression of H2BC9 in tumor and normal tissues was compared using the Wilcoxon rank-sum test. A paired-sample t test was performed to compare tumor tissues with paired adjacent normal tissues. The diagnostic values of H2BC9 were estimated using the diagnostic receiver operating characteristic (ROC) curves and all the area under curve (AUC) values were calculated. The relationship between clinicopathological features and H2BC9 expression was analyzed using the chi-square test. Cox regression analysis was conducted to investigate the influence of different variables on the prognosis of patients with HNSCC. For identifying risk factors impacting OS in HNSCC patients, univariate and multivariate Cox regression analyses were performed. All statistical analyses and visualizations were performed using the R software (version 4.2.1), SPSS (version 24.2; IBN, New York, USA), and GraphPad Prism (version 8.0). P < 0.05 was considered significantly different.

Results

Differences in H2BC9 expression between tumor and normal tissues

The abbreviations for the 20 TCGA cancer types are shown in Additional file 1: Table S1. H2BC9 expression was significantly upregulated in 14 of 20 tumor types in TCGA database: HNSC, LIHC, CHOL, KIRP, KIRC, LUAD, THCA, STAD, BRCA, ESCA, BLCA, CECS, LUSC, and UCEC (Fig. 1a). Further analysis of H2BC9 and HNSCC in both TCGA-HNSCC and GEO data revealed that their expression was significantly higher in tumor tissues than that in normal tissues (Fig. 1b–d). In 43 paired samples of tumors and adjacent tissues, H2BC9 was significantly upregulated in tumor tissues (P < 0.0001; Fig. 1e). ROC curve analysis demonstrated the diagnostic value of H2BC9 in distinguishing HNSCC tissues from normal tissues (AUC = 0.854, sensitivity = 0.81, specificity = 0.66) (Fig. 1f). This finding was corroborated by immunohistochemical results from the HPA database, showing increased H2BC9 expression in oral squamous cell carcinoma tissues compared with that in oral mucosa tissues (Fig. 1g). qRT-PCR and western blotting indicated that the relative mRNA and protein expression levels of H2BC9 were significantly elevated in NPC cell lines (C666-1 and 5-8F), laryngeal cancer cell lines (TU212 and Hep2), and the oral squamous cell carcinoma cell line (CAL-27) compared to normal head and neck epithelial cell line NP69 (Fig. 1h–j).

Fig. 1
figure 1

H2BC9 expression in patients with HNSCC. a H2BC9 expression levels in pan-cancer from TCGA database. b–d The expression of H2BC9 was significantly higher in HNSCC than that in normal tissue based on TCGA and GEO (GSE30784 and GSE51985) cohorts. e Expression of H2BC9 was significantly upregulated in tumors compared with that in paired normal tissues from the TCGA database. f Diagnostic ROC curves show the efficiency of H2BC9 in distinguishing tumors from non-tumor tissues. g The protein levels of H2BC9 in HNSCC tissues were higher than those in normal tissues in the Human Protein Atlas. h–j H2BC9 mRNA and protein expression levels were upregulated in HNSCC lines. Original blots presented in Additional file 2: Figure S1. a–d were analyzed using the Wilcoxon rank sum test, e was analyzed using the paired t test, and h–j were analyzed using the student’s t tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns not statistically significant. HNSCC: head and neck squamous cell carcinoma

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Relationship of H2BC9 expression and clinicopathologic characteristics in HNSCC

The association between H2BC9 mRNA expression and clinicopathological characteristics of HNSCC is presented in Table 1 and Fig. 2. High H2BC9 expression was correlated with advanced clinical stage, T stage, N stage and tissue type (all P < 0.05). However, no significant differences were observed in M stage, age, gender, histologic grade, and radiation therapy (Fig. 2a–i). Furthermore, H2BC9 expression varied by tumor type, with the lowest expression in tongue samples and the highest in mouth and throat samples (Fig. 2j).

Table 1 Clinical characteristics of the patients with HNSCC based on TCGA cohort
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Fig. 2
figure 2

Association between H2BC9 expression and clinicopathologic characteristics in HNSCC from TCGA. a Relationship between H2BC9 expression levels and clinicopathological features. b Clinical stage (stage I & stage II vs. stage III & stage IV). c T stage (T1 & T2 vs. T3 & T4). d N stage (N0 & N1 vs. N2 & N3). e M stage (M0 vs. M1). f Age (≤60 vs. >60). g Gender (female vs. male). h Histologic grade (G1 & G2 vs. G3 & G4). i Radiation therapy (no vs. yes). j H2BC9 expression in different tissues. a–i were analyzed using the Wilcoxon rank sum test, j was analyzed using Kruskal–Wallis rank sum test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns not statistically significant. T-tumor size status, N-lymph node status, M-metastasis, HNSCC: head and neck squamous cell carcinoma

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H2BC9 as an independent prognostic indicator for HNSCC. The prognostic value of H2BC9 in patients with HNSCC was first evaluated using the TCGA database. Kaplan–Meier analysis revealed that higher H2BC9 expression was associated with poorer OS (P = 0.017) and progression-free interval (PFI) (P = 0.048) (Fig. 3a, b). Time-dependent ROC curves demonstrated that H2BC9 had acceptable accuracy in predicting 1-year (AUC = 0.607), 3-year (AUC = 0.563), and 5-year survival rates (AUC = 0.588) (Fig. 3c). Univariate and multivariate Cox regression analyses identified H2BC9 expression (HR = 1.49; 95% CI = 1.04–2.14; P = 0.0301) and primary therapy outcome (HR = 0.2; 95% CI = 0.128–0.314; P < 0.01) as independent prognostic indicators for HNSCC (Fig. 3d, e). To validate the prognostic value of H2BC9, we performed a Kaplan–Meier analysis on clinical HNSCC samples from the First Affiliated Hospital of Guangxi Medical University. Elevated H2BC9 expression predicted a significantly poor 5-year OS (P < 0.001; Fig. 3f). Based on these findings, a nomogram was constructed incorporating age, primary therapy outcome, and H2BC9 expression to predict 1-, 3-, and 5-year survival probabilities (Fig. 3g). Calibration curves demonstrated that the predicted survival closely matched the actual outcomes, indicating strong predictive capabilities of the nomogram (Fig. 3h).

Fig. 3
figure 3

Prognostic significance of H2BC9 in HNSCC. a,b Kaplan–Meier curves of OS and PFI between low- and high- H2BC9 expression in patients with HNSCC. c Time-dependent ROC curve of the model. d Univariate Cox regression analysis of H2BC9 in HNSCC. e Multivariate Cox regression analysis of H2BC9 in HNSCC. f 5-year OS between high- and low-H2BC9 expression groups in clinical samples. g Nomogram integrating prognostic factors in HNSCC. h Calibration plot of the nomogram. OS: overall survival, PFI: progress-free interval, HNSCC: head and neck squamous cell carcinoma

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Function and pathway enrichment analysis

Differential expression analysis between high- and low-H2BC9 expression groups identified 283 DEGs, including 165 upregulated and 118 downregulated genes. The top 50 DEGs are presented in a heatmap (Fig. 4a). The GSEA of these DEGs showed significant enrichment in biological processes (BPs), cellular components (CCs), molecular functions (MFs), and various key pathways. Key BPs included regulation of mitotic nuclear division, DNA replication, and negative regulation of the immune response (Fig. 4b). Major CCs were nuclear chromosomes, spindles, and ribosomes (Fig. 4c). MFs included structural constituents of ribosomes, catalytic activity on RNA, mRNA binding, ATP-dependent activity on DNA, DNA helicase activity, and catalytic activity acting on DNA (Fig. 4d). Hallmark and KEGG pathway analyses indicated associations with E2F targets, Myc targets, G2M checkpoints, DNA repair, DNA replication, nucleotide excision repair, spliceosome, ribosome, and cell cycle pathways (Fig. 4e, f). Notably, DEGs linked to H2BC9 were involved in critical pathways and BPs related to tumorigenesis, including the cell cycle (NES = 2.28, P < 0.01), DNA replication (NES = 2.33, P < 0.01), and G2M checkpoint (NES = 2.69, P < 0.01) (Fig. 4g–i). Detailed enrichment analysis results are provided in Additional file 1: Table S2.

Fig. 4
figure 4

Cluster analysis of H2BC9 in TCGA HNSCC cohort. a Heat maps showing the correlation of the top 50 DEGs and H2BC9 expression. b–d GO term enrichment analysis of DEGs. e,f Hallmark and KEGG enrichment analysis of DEGs. g–i GSEA of DEGs. DEGs: differentially expressed genes, GO: Gene Ontology, KEGG: Kyoto Encyclopedia of Genes and Genomes, GSEA: gene set enrichment analysis, TCGA: The Cancer Genome Atlas, HNSCC: head and neck squamous cell carcinoma

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H2BC9 affects drug sensitivity in patients with HNSCC

To evaluate the relationship between H2BC9 expression and drug sensitivity in patients with HNSCC, we used a sensitivity score. We found that low H2BC9 expression significantly increased the sensitivity score for several drugs, including paclitaxel, docetaxel, cisplatin, 5-fluorouracil, gemcitabine, vinblastine, vinorelbine, AZD7762, MK-1775, tamoxifen, luminespib, Wee1 inhibitor, vincristine, AZD5438, nilotinib, and KRAS (G12C) inhibitor-12. This finding suggests that patients with low H2BC9 expression are more sensitive to these drugs (Fig. 5a–p). The remaining 62 drugs are listed in Additional file 1: Table S3. Conversely, high H2BC9 expression significantly increased sensitivity to doramapimod, ABT737, AZD6482, and nutlin-3a (−), indicating that these patients were more sensitive to these drugs (Fig. 5q–t).

Fig. 5
figure 5

Correlation between H2BC9 and drug sensitivity in HNSCC. a–t Drug sensitivity of patients with HNSCC with high- vs. low- H2BC9 expression. a–t were analyzed using the Wilcoxon rank sum test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, HNSCC: head and neck squamous cell carcinoma

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H2BC9 and its related genes expression in HNSCC cell lines

Using data from the DepMap database, we analyzed the correlation of H2BC9 expression with other genes in HNSCC cell lines. Key genes, such as TONSL, PITX2, NOTCH1, H2BC10, GAL, CGA, CCS, and APCS, were significantly positively correlated with H2BC9 expression (Fig. 6).

Fig. 6
figure 6

Correlation between H2BC9 and its related genes in HNSCC cell lines. a–h TONSL, PITX2, NOTCH1, H2BC10, GAL, CGA, CCS, and APCS. a–h were analyzed using Pearson’s correlation analyses. HNSCC: head and neck squamous cell carcinoma

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H2BC9 affects cell proliferation, cell cycle, apoptosis, and DNA damage in vitro

Our results indicate that H2BC9 is related to HNSCC prognosis and significantly enriched in the cell cycle pathway. Based on H2BC9 expression in HNSCC, we selected Cal-27 and Hep2 cell lines to explore the biological function of H2BC9. H2BC9 expression levels were significantly reduced after transfection with siRNA targeting H2BC9. As shown in Additional file 2: Figure S2. CCK-8 assay results demonstrated that the knockdown of H2BC9 significantly reduced the proliferation of HNSCC cells (Fig. 7a, b). Additionally, suppression of H2BC9 expression increased G2/M cell cycle arrest (Fig. 7c, d). Moreover, the downregulation of H2BC9 significantly enhanced apoptosis and DNA damage (Fig. 7e–g).

Fig. 7
figure 7

Knockdown of H2BC9 affects proliferation, cell cycle, apoptosis and DNA damage in vitro. a,b Knockdown of H2BC9 suppressed the proliferation of Cal-27 and Hep2 cells. c,d Knockdown of H2BC9 induced G2/M arrest of Cal-27 and Hep2 cells. e Knockdown of H2BC9 induced apoptosis of Cal-27 and Hep2 cells. f,g Knockdown of H2BC9 induced DNA damage of Cal-27 and Hep2 cells. Representative images of the comet assay and DNA damage in Cal-27 and Hep2 cells. Scale bars = 100 μm. Unpaired t tests and one-way or two-way analysis of variance were used to determine significance. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

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Discussion

The escalating prevalence of HNSCC continues to pose a persistent and formidable challenge to the realm of global public health [17,18,19]. The high diversity and heterogeneity of HNSCC make it arduous to identify key targets to develop effective cancer therapeutics and treatment strategies [20]. Identifying new biomarkers that accurately reflect the biological characteristics of HNSCC is crucial for improving disease detection and therapy. The oncogenesis process is inherently intricate and multifaceted, encompassing a spectrum of genetic alteration, epigenetic modification, and a dysregulated tumor microenvironment [21]. Histone protein alteration or abnormal expression can drive malignant transformation [22,23,24]. H2BC9 is involved in transcriptional dysregulation in cancer, cell cycle pathways, neutrophil degranulation, and interleukin signaling, all of which may play key roles in cancer progression [11]. However, the role of H2BC9 in HNSCC has been infrequently reported.

This study provides a comprehensive analysis of the predictive value of H2BC9 for stratifying clinicopathological characteristics in patients with HNSCC using a bioinformatics approach. We first utilized TCGA database to investigate H2BC9 expression across various cancers and found that H2BC9 was upregulated in 14 cancer types. Further analysis of TCGA and GEO databases revealed that H2BC9 expression was significantly higher in HNSCC tissues than in normal tissues in both paired and unpaired samples. ROC curves demonstrated the diagnostic value of H2BC9 in distinguishing patients with HNSCC from healthy individuals. Furthermore, high H2BC9 expression correlated with T-stage, N-stage, and overall clinical stage in HNSCC, suggesting its role in the development and progression of this cancer. qRT-PCR, western blotting, and IHC confirmed that H2BC9 was overexpressed in HNSCC cell lines and tissues, supporting the validity of our data.

To assess the prognostic significance of H2BC9 in patients with HNSCC, Kaplan–Meier curves were generated, showing that elevated H2BC9 expression was associated with poorer OS and PFI. A time-dependent ROC curve confirmed the prognostic efficiency of H2BC9 expression. Univariate and multivariate Cox regression analyses identified primary therapy outcomes and H2BC9 as independent prognostic indicators for patients with HNSCC. The constructed nomogram model accurately predicted the impact of age, primary therapy outcome, and H2BC9 expression on patient survival. Previous studies have shown that H2BC9 is an independent prognostic indicator for glioma and lung squamous cell carcinoma [9, 11]. In our study, the knockdown of H2BC9 expression reduced HNSCC cell proliferation, increased apoptosis, and induced DNA damage by arresting the G2/M cell cycle. Overall, these results suggest that H2BC9 is a potential diagnostic and prognostic biomarker for HNSCC.

In this study, we analyzed genes exhibiting abnormal expression patterns to understand the regulatory networks involving H2BC9 and the development of HNSCC. GSEA, combined with GO and KEGG enrichment analyses, revealed that H2BC9 is associated with the G2M checkpoint, DNA repair, DNA replication, nucleotide excision repair, spliceosome, ribosome, and cell cycle pathways. Current studies on H2BC9 in lung squamous cell carcinoma and glioma have mainly focused on its role in genetic alterations and cell cycle regulation [9, 11]. Cell cycle progression is crucial for cell proliferation and alteration and recognized as a hallmark of cancer [25, 26]. The G2/M checkpoints play an important role in cell cycle progression as they prevent DNA-damaged cells from entering mitosis [27]. In HNSCC, oncoprotein expression may cause gene mutations or inactivation, leading to a lack of Tp53 function and damage to G1/S cell cycle checkpoints, increasing the dependence on G2/M checkpoints [28,29,30]. Enrichment analysis indicated that the cell cycle, DNA replication, and G2/M checkpoints were significantly enriched in samples with high H2BC9 expression, suggesting a potential mechanism involving gene mutations and cell cycle damage resulting in G2/M checkpoint activation. We hypothesize that H2BC9 affects the cell cycle, leading to a poor prognosis of HNSCC. To confirm our hypothesis, we used siH2BC9 in cell experiments. The results showed that inhibiting H2BC9 expression caused cell cycle arrest in the G2/M phase while simultaneously enhancing apoptosis and DNA damage. However, further in vivo experiments are required to enhance the robustness of the results.

Recently, the importance of anticancer drug therapy has increased and drug therapy for HNSCC has rapidly advanced [31]. To better understand the effect of H2BC9 on drug sensitivity in HNSCC, we performed a correlation analysis. According to the National Comprehensive Cancer Network guidelines, chemotherapeutic agents, including paclitaxel, docetaxel, cisplatin, 5-FU, and gemcitabine, are commonly used as standard chemotherapy regimens for head and neck cancer [32]. Our analysis indicated that increased H2BC9 expression is associated with increased resistance to paclitaxel, docetaxel, carboplatin, 5-FU, cisplatin, and gemcitabine. This finding suggests that targeting H2BC9 expression may enhance the sensitivity of patients with HNSCC to these standard chemotherapeutic agents, improving treatment outcomes. Studies have reported that paclitaxel, docetaxel, and cisplatin can induce mitotic arrest or disrupt the cell cycle, specifically interrupting the G2/M phase during mitosis [33,34,35]. Our enrichment analysis suggests that upregulated H2BC9 is involved in activating G2/M checkpoints, potentially increasing DNA repair in cancer cells and allowing inappropriate proliferation [36]. The G2/M checkpoint is activated in response to endogenous DNA damage or damage induced by cancer treatments, including radiation therapy and cisplatin [37,38,39]. Therefore, we hypothesize that H2BC9 enhances G2/M checkpoint activation, influencing the sensitivity to these chemotherapeutic agents. Further research in vitro and in vivo experiments are required to validate this hypothesis.

Analysis of H2BC9 expression in HNSCC cell lines revealed that H2BC9 positively correlates with TONSL, PITX2, NOTCH1, H2BC10, GAL, CGA, CCS, and APCS. TONSL knockdown in ovarian cancer increases the G2/M cell cycle proteins, cyclin B and cyclin A, and the DNA break marker protein, γH2AX. Additionally, TONSL is often associated with copy number amplification (CNV) [40]. CNV is one of the most important classes of genomic mutations associated with carcinogenesis [41]. Our study found that the knockdown of H2BC9 leads to G2/M cycle arrest and DNA damage. Based on this, the intrinsic relationship between the influence of H2BC9 on the prognosis of patients with HNSCC, and gene copy number variation and cell cycle process interference will be a novel direction for subsequent research.

DNA methylation plays an important role in the etiology, pathogenesis and prognosis of HNSCC [42]. The investigation revealed that elevated expression levels of PITX2 correlate with suboptimal OS in HNSCC, as the hypermethylation of the PITX2 gene suppresses its protein expression [43]. In addition, methylation of GAL was associated with poor survival in patients with HPV-negative oropharyngeal cancer [44]. Members of the H2B family are hypermethylated in some tumors. For example, H2BC5, H2BC12, and H2BC18 were found to be hypermethylated in malignant pluripotent embryonic carcinoma, neuroblastomas, germ cell tumors, cervical squamous cell carcinoma, endometrial adenocarcinoma, and uterine carcinosarcoma [11]. In our study, high expression of H2BC9 is associated with poor prognosis in HNSCC, as a member of the H2B family, the methylation status of H2BC9 in the prognosis of HNSCC is a topic worth exploring.

NOTCH1 ranks as the second most frequently mutated gene, following closely behind TP53. Elevated NOTCH1 expression was correlated with an unfavorable prognostic outcome in HNSCC [45, 46]. HNSCC is one of the top five cancers with H2B mutations. Our investigation has elucidated a direct correlation between the expression levels of H2BC9 and the prognosis for patients with HNSCC, which requires further research.

The occurrence and development of tumors are intrinsically related to the changes of ubiquitin/ubiquitination (UB/UBL), which are widely involved in the control of the tumor cell cycle, apoptosis, and other biological processes [47]. In the OSCC prognostic model based on UB/UBL-related genes, H2BC10 was one of the high-risk factors [48]. Monoubiquitination of H2B may lead to chromosomal instability phenotypes associated with mitotic chromatin compression defects, which in turn promote tumorigenesis and progression [49]. In addition, H2BC10 is negatively correlated with CD8+ T cells, which is associated with increased pretumor immune invasion and high expression of immune checkpoints. The tumor immune microenvironment plays an important role in tumor progression, and H2BC9 is involved in tumor immune escape [50]. In this study, we found that H2BC9 affects the tumor cell cycle process and apoptosis. Therefore, whether this result is related to the changes of UB/UBL or the formation of immunosuppressive environment should be investigated.

Although CGA is rarely studied in HNSCC, the increased expression of CGA is involved in the occurrence of chemotherapy resistance in patients with gastric cancer through the CGA/EGFR/GATA2 pathway [51, 52]. Additionally, knocking out CCS in ovarian cancer cells increased cisplatin sensitivity. This may be mediated by the changes in SOD1 activity and ROS balance[53]. Our study found that patients with higher H2BC9 expression exhibited higher cisplatin sensitivity compared with that of patients with lower H2BC9 expression. However, the mechanism is still unclear. Further studies on the mechanism of H2BC9 interactions with these chemotherapy agents will help drug therapy decision-making. APCS, also known as SAP, has been poorly studied in HNSCC, but studies have shown that the absence of SPA can promote the progression of malignant insulinoma [54]. Therefore, understanding the relationship between H2BC9 and these genes may provide new insights into HNSCC research.

Although this research revealed some novel findings, it still has several limitations. First, the dataset employed in our investigation was predominantly sourced from a publicly accessible repository, the integrity of which is contingent upon rigorous evaluation and restricted to specific ethnic cohorts. Second, larger multi-center population cohorts were warranted to further confirm our findings. Third, in vitro and in vivo functional experiments are needed to further investigate the roles of H2BC9 in HNSCC initiation and progression, especially in cell cycle checkpoints and drug resistance.

Conclusion

Our results indicate that H2BC9 is a potential diagnostic and prognostic biomarker for HNSCC. H2BC9 may influence the progression of HNSCC and contribute to therapy resistance. These findings provide a basis for further investigation into the mechanisms by which H2BC9 affects HNSCC. Future research should focus on elucidating the specific molecular pathways regulated by H2BC9 and its role in therapeutic resistance. Additionally, understanding the interaction of H2BC9 with other biomarkers could lead to the development of more effective targeted therapies for HNSCC.

Availability of data and materials

The data underlying this article will be shared on reasonable request to the corresponding author.

Abbreviations

BP:

Biological process

CC:

Cellular component

DEG:

Differentially expressed gene

GO:

Gene Ontology

GSEA:

Gene set enrichment analysis

HNSCC:

Head and neck squamous cell carcinoma

HPA:

Human Protein Atlas

KEGG:

Kyoto Encyclopedia of Genes and Genomes

MF:

Molecular function

NOK:

Normal oral keratinocytes

NPC:

Nasopharyngeal carcinoma

OS:

Overall survival

PI:

Propidium iodide

PFI:

Progression-free interval

qRT-PCR:

Quantitative real-time PCR

TCGA:

The Cancer Genome Atlas

TPM:

Transcripts per million

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Acknowledgements

We thank Mr. Jindong Zhu (Anrenxin Biotechnology Co, Ltd at Nanning, China) for his helpful discussions and bioinformatics analysis.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 82202940, 82272736, 82160467, 81460460, 81760542), The Research Foundation of the Science and Technology Department of Guangxi Province, China (grant No. 2023GXNSFDA026009, 2016GXNSFAA380252, 2018AB61001 and 2014GXNSFBA118114), the Research Foundation of the Health Department of Guangxi Province, China (No. S2018087), Guangxi Medical University Training Program for Distinguished Young Scholars (2017), Medical Excellence Award Funded by the Creative Research Development Grant from the First Affiliated Hospital of Guangxi Medical University (2016). Guangxi Medical High-level Talents Training Program, the central government guide local science and technology development projects (ZY18057006) and the Joint Project on Regional High-Incidence Diseases Research of the Guangxi Natural Science Foundation (grant no. 2023GXNSFBA026211.

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Author notes

  1. Lanhua Wu and Liang Li have contributed equally to this work.

Authors and Affiliations

  1. Department of Radiation Oncology, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, Guangxi, China

    Lanhua Wu, Mingjing Zhu, Min Kang & Li Jiang

  2. Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor (Guangxi Medical University) , Ministry of Education, Nanning, 530021, Guangxi, China

    Lanhua Wu, Mingjing Zhu, Min Kang & Li Jiang

  3. Department of Toxicology, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China

    Lanhua Wu, Yueming Jiang, Min Kang & Li Jiang

  4. Guangxi Colleges and Universities Key Laboratory of Prevention and Control of Highly Prevalent Diseases, Guangxi Medical University, Nanning, China

    Lanhua Wu, Yueming Jiang, Min Kang & Li Jiang

  5. CPC Organization and Human Resource Department, The First Affiliated Hospital of Guangxi Medical University, Nanning, 530021, Guangxi, China

    Liang Li

  6. Guangdong Cardiovascular Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China

    Ziyan Zhou

  7. Department of Oncology, Liuzhou People’s Hospital Affiliated to Guangxi Medical University, Liuzhou, 545006, Guangxi, China

    Xuejin Su

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Contributions

LJ conceived and designed the project, MK designed the project and gave important suggestions to the manuscript, LHW acquired the data and written the original draft, LL analysed and interpreted the data and written the original draft, MJZ, ZYZ, XJS, and YMJ reviewed and edited draft. All the authors contributed to the manuscript and approved the submitted version.

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Correspondence to Min Kang or Li Jiang.

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Ethical approval and consent to participate

The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University and the approval number was 2024-E533-01. This study has been officially sanctioned by the Medical Ethics Committee of the First Affiliated Hospital of Guangxi Medical University, thereby waiving the requirement for informed consent from participants. The study did not involve minors. All experiments were performed in accordance with relevant guidelines and regulations. The study was conducted according to the Declaration of Helsinki.

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Supplementary Information

Additional file 1: Table S1

: Abbreviations of the 20 cancer types in The Cancer Genome Atlas database. Table S2: The enrichment analysis of H2BC9 differentially ex-pressed genes. Table S3: Drug sensitivity of H2BC9 in HNSCC

Additional file 2: Figure S1

: Original western blotting results. (a) GAPDH. (b) H2BC9. Figure S2: Construction of HNSCC cell models with H2BC9 knockdown. (a, b) qPCR were applied to examine the knockdown efficiency of H2BC9 in Cal-27 and Hep2. Unpaired two-tailed Student’s t-tests were used to determine significance. ***P < 0.001

Additional file 3:

The raw data sources of this study

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Wu, L., Li, L., Zhu, M. et al. Evaluating H2BC9 as a potential diagnostic and prognostic biomarker in head and neck squamous cell carcinoma. Eur J Med Res 30, 54 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40001-025-02301-3

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European Journal of Medical Research

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