Deregulation of MMP-2 and MMP-9 in laryngeal cancer: A retrospective observational study.

Laryngeal carcinoma (LC) is reported to have a higher incidence rate among all types of head and neck cancers around the globe. Mechanisms resulting in the pathogenesis of LC are complicated due to involvement of invasion and metastasis and there is a need to understand this complicated multistep process. Numerous molecules including matrix metalloproteinases (MMPs) are involved in regulating metastatic mechanisms. Furthermore, activation and expression of different classes of MMPs have been observed in multiple pathological and physiological events including inflammation, invasion, and metastasis. Among all members of MMPs, matrix metalloproteinases-2 (MMP-2), and matrix metalloproteinases-9 (MMP-9) have been frequently reported to correlate with tumor pathogenesis. The present study is designed to check the involvement of MMP-2 and MMP-9 in LC pathogenesis. 184 laryngeal tumor samples along with adjacent uninvolved healthy sections were collected to check the expression deregulation of the above-mentioned gene in LC using real-time PCR and immunohistochemistry (IHC). Real-time PCR and IHC analyses showed the significant upregulation of MMP-2 (P < .0001) and MMP-9 (P < .0001) genes in laryngeal tumors compared to controls. Spearman correlation showed the positive correlation of expression deregulation of selected MMPs with advanced TNM stage [MMP-2, (P < .0001); MMP-9, P < .0001] and smoking status [MMP-2 (P < .0001); MMP-9 P < .0001] in laryngeal pathogenesis. Receiver operating curve (ROC) analysis showed the good diagnostic/prognostic value of said markers in laryngeal cancer patients. The present study showed that significant upregulation of selected MMPs was found associated with an increased risk of laryngeal cancer and can act as good diagnostic markers for the detection of said disease.

LncRNA NEAT1 promotes proliferation, migration, and invasion of laryngeal squamous cell carcinoma cells through miR-411-3p/FZD3-mediated Wnt signaling pathway.

The lncRNA NEAT1 has been shown to promote the progression of several cancers, containing laryngeal squamous cell carcinoma (LSCC). However, the precise mechanism by which it promotes LSCC progression remains unclear. In this study, we verified the high expression of lncRNA NEAT1 in LSCC tissues and cells using RT-qPCR. Analysis of clinical data exhibited that high expression of lncRNA NEAT1 was associated with a history of smoking, worse T stage, lymph node metastasis, and later TNM stage in patients with LSCC. The promotion effect of lncRNA NEAT1 on LSCC cell proliferation, migration, invasion, and tumor growth in vivo was verified by CCK-8, plate clone formation, Transwell, and nude mouse tumorigenicity assays. Bioinformatics prediction and double luciferase reporter gene assay verified the binding of miR-411-3p to lncRNA NEAT1 and FZD3 mRNA, and inhibition of miR-411-3p reversed the inhibitory effect of lncRNA NEAT1 on FZD3 expression in LSCC cells. We also verified that lncRNA NEAT1-mediated FZD3 activation in the Wnt pathway affects LSCC development. In conclusion, we demonstrate that lncRNA NEAT1 promotes the progression of LSCC, and propose that the lncRNA NEAT1/miR-411-3p/FZD3 axis may be an effective target for LSCC therapy.

LncRNA PCAT6 promotes the occurrence of laryngeal squamous cell carcinoma via modulation of the miR-4731-5p/NOTCH3 axis.

Laryngeal squamous cell carcinoma (LSCC) is one of the most aggressive cancers that affect the head and neck region. Recent researches have confirmed that long non-coding RNAs (lncRNAs) present an emerging role in diversiform diseases including cancers. Prostate cancer-associated ncRNA transcript 6 (PCAT6) is an oncogene in lung cancer, cervical cancer, colon cancer and gastric cancer, but its role in LSCC is still unknown. In the current study, we attempted to figure out the role of PCAT6 in LSCC. RT-qPCR was to analyze PCAT6 expression in LSCC cells. Functional assays were to uncover the role of PCAT6 in LSCC. Mechanism assays were to explore the regulatory mechanism behind PCAT6 in LSCC. PCAT6 exhibited higher expression in LSCC cells and PCAT6 strengthened cell proliferation and inhibited cell apoptosis. Furthermore, lncRNA PCAT6 modulated notch receptor 3 expression and activated NOTCH signaling pathway via serving as a sponge for miR-4731-5p. Taken together, lncRNA PCAT6 was identified as an oncogene in LSCC, which revealed that PCAT6 might be used as potential therapeutic target for LSCC.

Epigenetic mechanism of RBM15 in affecting cisplatin resistance in laryngeal carcinoma cells by regulating ferroptosis.

Laryngeal carcinoma (LC) is a common cancer of the respiratory tract. This study aims to investigate the role of RNA-binding motif protein 15 (RBM15) in the cisplatin (DDP) resistance of LC cells. LC-DDP-resistant cells were constructed. RBM15, lysine-specific demethylase 5B (KDM5B), lncRNA Fer-1 like family member 4 (FER1L4), lncRNA KCNQ1 overlapping transcript 1 (KCNQ1OT1), glutathione peroxidase 4 (GPX4), and Acyl-CoA synthetase long-chain family (ACSL4) was examined. Cell viability, IC50, and proliferation were assessed after RBM15 downregulation. The enrichment of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) and N6-methyladenosine (m6A) on KDM5B was analyzed. KDM5B mRNA stability was measured after actinomycin D treatment. A tumor xenograft assay was conducted to verify the role of RBM15 in LC. Results showed that RBM15 was upregulated in LC and its knockdown decreased IC50, cell viability, proliferation, glutathione, and upregulated iron ion content, ROS, malondialdehyde, ACSL4, and ferroptosis. Mechanistically, RBM15 improved KDM5B stability in an IGF2BP3-dependent manner, resulting in FER1L4 downregulation and GPX4 upregulation. KDM5B increased KCNQ1OT1 and inhibited ACSL4. KDM5B/KCNQ1OT1 overexpression or FER1L4 knockdown promoted DDP resistance in LC by inhibiting ferroptosis. In conclusion, RBM15 promoted KDM5B expression, and KDM5B upregulation inhibited ferroptosis and promoted DDP resistance in LC by downregulating FER1L4 and upregulating GPX4, as well as by upregulating KCNQ1OT1 and inhibiting ACSL4. Silencing RBM15 inhibited tumor growth in vivo.

Identification and bioinformatic characterization of a serum miRNA signature for early detection of laryngeal squamous cell carcinoma.

BACKGROUND: The growing understanding of cancer biology and the establishment of new treatment modalities has not yielded the expected results in terms of survival for Laryngeal Squamous Cell Cancer (LSCC). Early diagnosis, as well as prompt identification of patients with high risk of relapse would ensure greater chance of therapeutic success. However, this goal remains a challenge due to the absence of specific biomarkers for this neoplasm. METHODS: Serum samples from 45 LSCC patients and 23 healthy donors were collected for miRNA expression profiling by TaqMan Array analysis. Additional 20 patients and 42 healthy volunteers were included for the validation set, reaching an equal number of clinical samples for each group. The potential diagnostic ability of the such identified three-miRNA signature was confirmed by ROC analysis. Moreover, each miRNA was analyzed for the possible correlation with HNSCC patients' survival and TNM status by online databases Kaplan-Meier (KM) plotter and OncomiR. In silico analysis of common candidate targets and their network relevance to predict shared biological functions was finally performed by PANTHER and GeneMANIA software. RESULTS: We characterized serum miRNA profile of LSCC patients identifying a novel molecular signature, including miR-223, miR-93 and miR-532, as circulating marker endowed with high selectivity and specificity. The oncogenic effect and the prognostic significance of each miRNA was investigated by bioinformatic analysis, denoting significant correlation with OS. To analyse the molecular basis underlying the pro-tumorigenic role of the signature, we focused on the simultaneously regulated gene targets-IL6ST, GTDC1, MAP1B, CPEB3, PRKACB, NFIB, PURB, ATP2B1, ZNF148, PSD3, TBC1D15, PURA, KLF12-found by prediction tools and deepened for their functional role by pathway enrichment analysis. The results showed the involvement of 7 different biological processes, among which inflammation, proliferation, migration, apoptosis and angiogenesis. CONCLUSIONS: In conclusion, we have identified a possible miRNA signature for early LSCC diagnosis and we assumed that miR-93, miR-223 and miR-532 could orchestrate the regulation of multiple cancer-related processes. These findings encourage the possibility to deepen the molecular mechanisms underlying their oncogenic role, for the desirable development of novel therapeutic opportunities based on the use of short single-stranded oligonucleotides acting as non-coding RNA antagonists in cancer.

Identification of a Novel Five-Gene Prognostic Model for Laryngeal Cancer Associated with Mitophagy Using Integrated Bioinformatics Analysis and Experimental Verification.

Laryngeal cancer (LC) is a prevailing tumor with a high mortality rate. The pivotal role of mitophagy in LC is acknowledged; however, a comprehensive analysis of the corresponding genes has not been conducted. In the present study, we proposed a prognostic model consisting of mitophagy-related genes in LC. Clinical information and transcriptome profiling of patients with LC and mitophagy-related genes were retrieved from open-source databases. Gene set variation analysis (GSVA) and Weighted Gene Co-expression Network Analysis (WGCNA) were used to identify core mitophagy-related genes and construct gene co-expression networks. Functional enrichment analysis was employed to analyze the enriched regulatory pathways of the mitophagy-related genes. Kaplan-Meier curves (KM), Cox, and LASSO regression were applied to explore their prognostic effects. Finally, quantitative real-time PCR (RT-qPCR) further verified the bioinformatics prediction. A total of 45 genes related to mitochondrial pathways was collected. GSVA analysis demonstrated that these genes in tumor samples mainly referred to the mitochondrial pathway. Among these genes, five mitophagy-related-gene signatures (CERCAM, CHPF, EPHX3, EXT2, and MED15) were further identified to construct the prognostic model. KM and Cox regression analyses indicated that this model had an accurate prognostic prediction for LC. RT-qPCR showed that CERCAM, CHPF, EXT2, and MED15 expression were upregulated, and EPHX3 level was decreased in LC cells. The present study established a five-mitophagy-related-gene model that can predict the prognosis of LC patients, thus laying the foundation for a better understanding and potential advancements in clinical treatments for LC.

Exploring the protective association between COVID-19 infection and laryngeal cancer: insights from a Mendelian randomization study.

Introduction: Viral infections have been implicated as a risk factor for laryngeal cancer. Given the possible effects of Corona virus disease 2019 (COVID-19) on the laryngeal tissue, we investigated the causal link between COVID-19 and laryngeal cancer using a two-sample Mendelian randomization (MR) approach. Methods: We utilized genetic data from the 5th Genome-wide association studies (GWAS) edition of the COVID-19 Host Genetics Initiative (published on January 18, 2021) and a large-scale laryngeal cancer GWAS comprising 180 cases and 218,612 controls of European ancestry. We applied inverse variance weighting, MR Egger, and weighted median methods to infer causality. We performed sensitivity analysis using the "leave-one-out" method to verify robustness. Results: We found no evidence of a causal association between gene-predicted COVID-19 and laryngeal cancer [Odds ratio (OR)=0.24 (95% Confidence intervals (CI), 0.05-1.26), P=0.09]. However, we observed significant inverse associations between gene-predicted COVID-19 hospitalization [OR=0.51 (95% CI, 0.28-0.95), P=0.03] and severe patients [OR=0.62 (95% CI, 0.43-0.90), P=0.01] and laryngeal cancer. Notably, the study detected important genetic variants, such as rs13050728, that modulate the expression of interferon alpha receptor 2 (IFNAR2), indicating possible roles for immune response pathways in both COVID-19 and cancer. Discussion: This study reveals a potential interaction between COVID-19 severity, genetic factors, and laryngeal cancer, underscoring the importance of investigating the immune response mechanisms in both conditions. These findings contribute to the understanding of the complex interactions between COVID-19 and laryngeal cancer and may guide future research on the role of immune response, particularly involving IFNAR2.

DIAPH2 gene polymorphisms and laryngeal cancer risk in men.

BACKGROUND: The DIAPH2 gene is one of the genes commonly associated with laryngeal squamous cell carcinoma (LSCC). In our study, we considered the four polymorphisms of this gene, i.e. rs5920828, rs4322175, rs12851931 and rs5921830 as potential genetic risk factors for LSCC. METHODS: We determined the genotyping of the genetic variants of DIAPH2 in 230 male patients with histologically confirmed LSCC compared to the European population. Demographic and environmental exposure data of each subject were examined. To conduct the genetic tests, extraction of total DNA was performed. We genotyped all four variants in each patient and determined their frequencies. RESULTS: In the case of the rs12851931 polymorphism in the DIAPH2 gene, a significant difference was observed in the distribution of the T stage depending on the polymorphism. Heterozygotes were more often associated with T2 stage, while homozygotes were more likely to have higher tumor stages. The rs12851931 homozygotes of DIAPH2 were statistically significantly more prevalent in smokers. The results suggested that rs12851931 polymorphism in DIAPH2 could increase the onset risk of LSCC. CONCLUSIONS: Our results provide further information on the role of the DIAPH2 gene in the pathogenesis of LSCC.

Identification of Regulatory RNA-Binding Proteins Associated with Immune Infiltration in Laryngeal Squamous Cell Carcinoma.

We analyzed bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) data to identify alternative splicing (AS) events and regulatory RNA-binding proteins (RBPs) associated with immune infiltration in human laryngeal squamous cell carcinoma (LSCC). Whole-transcriptome sequencing data of 20 human laryngeal cancer and paracancerous tissues were downloaded from the Gene Expression Omnibus public database, using newly published splicing-site usage variation analysis software to obtain highly conserved regulated AS (RAS) events, and scientific reverse convolution algorithm analysis was used to identify significantly different immune cells and perform a correlation analysis between the two. The software package edgeR was used to identify differentially expressed RBPs and the immune infiltration-related LSCC-RAS they may regulate. Finally, we present the expression profiles and survival curves of 117 human laryngeal cancer samples from The Cancer Genome Atlas dataset for the identified RBPs and LSCC-RAS. We also downloaded the gene set enrichment 150321 scRNA-seq data for two human LSCC tissue samples. The RBP expression pattern and the expression of prophase RBP genes were analyzed in different LSCC cell populations. RNA-binding motif protein 47 (RBM47) and filamin A, as well as the RBP-RAS events that were screened in both the fibulin 2 and fibronectin 1 genes, were all significantly associated with the prognosis, and the RBM47 gene was upregulated in myeloid cells. Because the prognosis was significantly associated with two RBP regulators and two LSCC-RAS events, they may be critical regulators of immune cell survival during laryngeal cancer progression, and RBM47 may regulate macrophage-associated AS and affect immunity.

The Relationship between miR-5682 and Nutritional Status of Radiotherapy-Treated Male Laryngeal Cancer Patients.

BACKGROUND: Nutritional deficiencies are frequently observed in patients with head and neck cancer (HNC) undergoing radiation therapy. microRNAs (miRNAs) were found to play an important role in the development of metabolic disorders throughout regulation of genes involved in inflammatory responses. This study aimed to explore the correlation between pre-treatment miR-5682 expression and parameters reflecting nutritional deficits in laryngeal cancer (LC) patients subjected to radiotherapy (RT). METHODS: Expression of miR-5682 was analyzed in plasma samples of 56 male LC individuals. Nutritional status of LC patients was assessed using anthropometric and laboratory parameters, bioelectrical impedance analysis (BIA) and clinical questionnaires. RESULTS: A high expression of miR-5682 was associated with significantly lower values of BMI, fat mass, fat-free mass and plasma albumin at selected periods of RT course. miR-5682 allowed us to distinguish between patients classified with both SGA-C and low albumin level from other LC patients with 100% sensitivity and 69.6% specificity (AUC = 0.820; p < 0.0001). Higher expression of studied miRNA was significantly associated with shorter median overall survival (OS) in LC patients (HR = 2.26; p = 0.008). CONCLUSIONS: analysis of miR-5682 expression demonstrates a potential clinical utility in selection of LC patients suffering from nutritional deficiencies developing as a consequence of RT-based therapy.

Effects of VDR and CYP24A1 gene polymorphisms on the outcome of supraglottic larynx cancer.

OBJECTIVE: Vitamin D has been demonstrated to play a protective role in carcinogenesis. Polymorphisms of the vitamin D receptor (VDR) genes and 24-α-hydroxylase (encoded by CYP24A1) may affect the outcome of some cancers. This study examines the effects of the VDR gene and CYP24A1 single nucleotide polymorphisms on the outcome of supraglottic larynx cancer. PATIENTS AND METHODS: Patients diagnosed with supraglottic larynx cancer between 2017 and 2022 were enrolled. Single nucleotide polymorphisms of the VDR gene (rs2228570, rs731236, rs7975232, rs11574113, rs11168267 and rs11168266) and CYP24A1 gene (rs4809960, rs6022999, rs6068816, rs2259735 and rs2296241) were investigated. All patients were followed up for any evidence of local recurrence, regional recurrence, distant metastasis, and second primary tumor development. Cox regression analysis was performed to evaluate the prognostic value of single-nucleotide polymorphisms (SNPs). Kaplan-Meier method was used for survival analysis. RESULTS: 87 patients were included. The mean follow-up time was 45.02±24.47 months. Cox regression analysis for locoregional recurrence revealed that the hazard ratio of rs731236 GG was 2.098 (95% CI, range: 1.047-4.202, p=0.037). Locoregional recurrence for rs731236 AA, AG, and GG were 38.6%, 23.1%, and 53.3%, respectively. In the presence of rs731236 GG polymorphism, disease-specific survival was significantly shorter (47.63±7.48 months, p=0.015), and disease-free survival (45.71±6.3 months) was significantly shorter (p=0.040). Rates of metastases and second primary tumors were not significantly different between SNPs. CONCLUSIONS: This study has demonstrated the possible effects of VDR rs731236 SNP on the locoregional recurrence and prognosis of supraglottic larynx cancer.

Peritumor Mucosa in Advanced Laryngeal Carcinoma Exhibits an Aberrant Proangiogenic Signature Distinctive from the Expression Pattern in Adjacent Tumor Tissue.

The field cancerization theory is an important paradigm in head and neck carcinoma as its oncological repercussions affect treatment outcomes in diverse ways. The aim of this study is to assess the possible interconnection between peritumor mucosa and the process of tumor neoangiogenesis. Sixty patients with advanced laryngeal carcinoma were enrolled in this study. The majority of patients express a canonical HIF-upregulated proangiogenic signature with almost complete predominancy of HIF-1α overexpression and normal expression levels of the HIF-2α isoform. Remarkably, more than 60% of the whole cohort also exhibited an HIF-upregulated proangiogenic signature in the peritumoral benign mucosa. Additionally, the latter subgroup had a distinctly shifted phenotype towards HIF-2α upregulation compared to the one in tumor tissue, i.e., a tendency towards an HIF switch is observed in contrast to the dominated by HIF-1α tumor phenotype. ETS-1 displays stable and identical significant overexpression in both the proangiogenic phenotypes present in tumor and peritumoral mucosa. In the current study, we report for the first time the existence of an abnormal proangiogenic expression profile present in the peritumoral mucosa in advanced laryngeal carcinoma when compared to paired distant laryngeal mucosa. Moreover, we describe a specific phenotype of this proangiogenic signature that is significantly different from the one present in tumor tissue as we delineate both phenotypes, quantitively and qualitatively. This finding is cancer heterogeneity, per se, which extends beyond the "classical" borders of the malignancy, and it is proof of a strong interconnection between field cancerization and one of the classical hallmarks of cancer-the process of tumor neoangiogenesis.

HOXB9 promotes laryngeal squamous cell carcinoma progression by upregulating MMP12.

Transcriptional factor HOXB9, a part of the HOX gene family, plays a crucial role in the development of diverse cancer types. This study aimed to elucidate the regulatory mechanism of HOXB9 on the proliferation and invasion of laryngeal squamous cell carcinoma (LSCC) cells to provide guidance for the development and prognosis of LSCC. The CRISPR/Cas9 method was employed in LSCC cell lines to knock out the HOXB9 gene and validate its effects on the proliferation, migration, invasion, and regulation of LSCC cells. CCK-8 and flow cytometry were used to detect cell viability and proliferation; Tunnel was used to detect cell apoptosis, and transwell was used to detect cell migration and invasion. The effect of HOXB9 on tumor growth was tested in nude mice. The downstream target genes regulated by HOXB9 were screened by microarray analysis and verified by Western blotting, immunohistochemistry, chromatin immunoprecipitation, and double-luciferase reporter assays. The current research investigated molecular pathways governed by HOXB9 in the development of LSCC. Additionally, both laboratory- and living-organism-based investigations revealed that disrupting the HOXB9 gene through the CRISPR/CAS9 mechanism restrained cellular growth, movement, and infiltration, while enhancing cellular apoptosis. Detailed analyses of LSCC cell strains and human LSCC samples revealed that HOXB9 promoted LSCC progression by directly elevating the transcriptional activity of MMP12. HOXB9 could influence changes in LSCC cell functions, and the mechanism of action might be exerted through its downstream target gene, MMP12.

Comprehensive analysis of the immune implication of EPHX4 gene in laryngeal squamous cell carcinoma.

OBJECTIVES: The role of Epoxide Hydrolase-4 (EPHX4), a member of epoxide hydrolase family, has not been investigated in cancer. The purpose of this article is to explore the application value of EPHX4 in laryngeal cancer and its relationship with immune infiltration. METHODS: We observed that EPHX4 expression and its survival assays in laryngeal cancer specimens based on The Cancer Genome Atlas (TCGA) cohorts. We also analyzed the correlation between immune cell infiltration levels and EPHX4 gene copy number in laryngeal cancer. Finally, we conducted in vitro assay to evaluate the functions of EPHX4 in laryngeal cancer cell line. RESULTS: EPHX4 is highly expressed in laryngeal cancer specimens and has a poor prognosis. EPHX4 related immune cell analysis showed that it participated in NK Natural killer cell mediated cytotoxicity. Finally, Cell experiments indicate that EPHX4 could promote laryngeal cancer cell line proliferation, colony formation and invasion. CONCLUSIONS: Our research results suggest that EPHX4 may be a potential immunotherapy target for laryngeal cancer. The nominated immune signature is a helpful and promising prognostic indicator in laryngeal cancer. LEVELS OF EVIDENCE: Level 3.

Oridonin inhibited epithelial-mesenchymal transition of laryngeal carcinoma by positively regulating LKB1/AMPK signaling.

Oridonin is the main bioactive component of Rabdosia rubescens, and its anticancer activity has been reported in a variety of cancers. However, the molecular mechanism of oridonin in laryngeal carcinoma remains unclear. In the present study, the cytotoxic effect of oridonin on laryngeal carcinoma Hep-2 and TU212 cell lines were initially detected by modified MTT assay. The results showed that oridonin had a dose-dependent anti-proliferative effect on laryngeal carcinoma Hep-2 and TU212 cells. Next, we found that oridonin significantly inhibited the migration and invasion of human laryngeal carcinoma Hep-2 and TU212 cell lines by wound healing assay and transwell assay. Subsequently, the results of quantitative real-time PCR assay and western blotting assay confirmed that oridonin upregulated the expression of E-cadherin while downregulated the expression of N-cadherin in a concentration-dependent manner at mRNA and protein levels. In addition, phosphorylation levels of liver kinase B1 (p-LKB1) and AMP-activated protein kinase (p-AMPK) were also elevated upon oridonin treatment. To further verify the role of LKB1/AMPK signaling pathway in laryngeal carcinoma, overexpression of LKB1 was constructed by plasmid transfection. The data exhibited that overexpression of LKB1 could further reinforce the increase of E-cadherin level and decrease of N-cadherin level mediated by oridonin. Additionally, AMPK inhibitor compound C could reverse anti-metastatic effect of oridonin on laryngeal carcinoma, and antagonise EMT expression. In contrast, AMPK activator AICAR presented the opposite effect. In conclusion, our study revealed that oridonin could remarkably reverse the epithelial-mesenchymal transition of laryngeal carcinoma by positively regulating LKB1/AMPK signaling pathway, which suggested that oridonin may be a potential candidate for the treatment of laryngeal carcinoma in the future.

Combined PIK3CA and SOX2 Gene Amplification Predicts Laryngeal Cancer Risk beyond Histopathological Grading.

The PIK3CA and SOX2 genes map at 3q26, a chromosomal region frequently amplified in head and neck cancers, which is associated with poor prognosis. This study explores the clinical significance of PIK3CA and SOX2 gene amplification in early tumorigenesis. Gene copy number was analyzed by real-time PCR in 62 laryngeal precancerous lesions and correlated with histopathological grading and laryngeal cancer risk. Amplification of the SOX2 and PIK3CA genes was frequently detected in 19 (31%) and 32 (52%) laryngeal dysplasias, respectively, and co-amplification in 18 (29%) cases. The PIK3CA and SOX2 amplifications were predominant in high-grade dysplasias and significantly associated with laryngeal cancer risk beyond histological criteria. Multivariable Cox analysis further revealed PIK3CA gene amplification as an independent predictor of laryngeal cancer development. Interestingly, combined PIK3CA and SOX2 amplification allowed us to distinguish three cancer risk subgroups, and PIK3CA and SOX2 co-amplification was found the strongest predictor by ROC analysis. Our data demonstrate the clinical relevance of PIK3CA and SOX2 amplification in early laryngeal tumorigenesis. Remarkably, PIK3CA amplification was found to be an independent cancer predictor. Furthermore, combined PIK3CA and SOX2 amplification is emerging as a valuable and easy-to-implement tool for cancer risk assessment in patients with laryngeal precancerous lesions beyond current WHO histological grading.

Identification of Down-Expressed CRNN Associated with Cancer Progression and Poor Prognosis in Laryngeal Squamous Cell Carcinoma.

BACKGROUND: The prevalence of laryngeal squamous cell carcinoma (LSCC) is increasing, and it poses a significant threat to human health; therefore, identifying specific targets for LSCC remains crucial. METHODS: Bioinformatics analysis was used to compare the different expression genes expressed in LSCC. Immunohistochemical assay and western blotting were used to analysis protein expression. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)((4,5 Dimethyl thiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide)4,5 Dimethyl thiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) and 5-ethynyl 2'-deoxyuridine (Edu) assay. Flow cytometry was used to measure the cell cycle. Cell migration was measured by wound healing assay and transwell assay. RESULTS: Our analysis revealed 36 upregulated and 65 downregulated differentially expressed genes (DEGs) when comparing LSCC tumors to adjacent tissues, with cornulin (CRNN) identified as a key hub gene connecting these DEGs. We observed a consistent downregulation of CRNN expression in LSCC cell lines and tissues and was associated with poor patient survival and the tumor microenvironment. CRNN overexpression was found to significantly inhibit cell growth, cell cycle progression, migration and invasion, while CRNN knockdown had the opposite effects. Additionally, in vivo experiments demonstrated that CRNN overexpression suppressed tumor growth in nude mice. CONCLUSIONS: CRNN functions as a potential tumor suppressor and regulates important aspects of LSCC, providing valuable insights into the role of CRNN in LSCC pathogenesis and potential for targeted therapeutic interventions.

BNIPL is a promising biomarker of laryngeal cancer: novel insights from bioinformatics analysis and experimental validation.

BACKGROUND: Laryngeal cancer (LC) is a malignant tumor with high incidence and mortality. We aim to explore key genes as novel biomarkers to find potential target of LC in clinic diagnosis and treatment. METHODS: We retrieved GSE143224 and GSE84957 datasets from the Gene Expression Omnibus database to screen the differentially expressed genes (DEGs). Hub genes were identified from protein-protein interaction networks and further determined using receiver operating characteristic curves and principal component analysis. The expression of hub gene was verified by quantitative real time polymerase chain reaction. The transfection efficiency of BCL2 interacting protein like (BNIPL) was measured by western blot. Proliferation, migration, and invasion abilities were detected by Cell Counting Kit-8, wound-healing, and transwell assays, respectively. RESULTS: Total 96 overlapping DEGs were screened out from GSE143224 and GSE84957 datasets. Six hub genes (BNIPL, KRT4, IGFBP3, MMP10, MMP3, and TGFBI) were identified from PPI network. BNIPL was selected as the target gene. The receiver operating characteristic curves of BNIPL suggested that the false positive rate was 18.5% and the true positive rate was 81.5%, showing high predictive values for LC. The expression level of BNIPL was downregulated in TU212 and TU686 cells. Additionally, overexpression of BNIPL suppressed the proliferation, migration, and invasion of TU212 and TU686 cells. CONCLUSION: BNIPL is a novel gene signature involved in LC progression, which exerts an inhibitory effect on LC development. These findings provide a novel insight into the pathogenesis of LC.

m6A/HOXA10-AS/ITGA6 axis aggravates oxidative resistance and malignant progression of laryngeal squamous cell carcinoma through regulating Notch and Keap1/Nrf2 pathways.

As the second most prevalent malignant tumor of head and neck, laryngeal squamous cell carcinoma (LSCC) imposes a substantial health burden on patients worldwide. Within recent years, resistance to oxidative stress and N6-methyladenosine (m6A) of RNA have been proved to be significantly involved in tumorigenesis. In current study, we investigated the oncogenic role of m6A modified long non coding RNAs (lncRNAs), specifically HOXA10-AS, and its downstream signaling pathway in the regulation of oxidative resistance in LSCC. Bioinformatics analysis revealed that heightened expression of HOXA10-AS was associated with the poor prognosis in LSCC patients, and N (6)-Methyladenosine (m6A) methyltransferase-like 3 (METTL3) was identified as a factor in promoting m6A modification of HOXA10-AS and further intensify its RNA stability. Mechanistically, HOXA10-AS was found to play as a competitive endogenous RNA (ceRNA) by sequestering miR-29 b-3p and preventing its downregulation of Integrin subunit alpha 6 (ITGA6), ultimately enhancing the oxidative resistance of tumor cells and promoting the malignant progression of LSCC. Furthermore, our research elucidated the mechanism by which ITGA6 accelerates Keap1 proteasomal degradation via enhancing TRIM25 expression, leading to increased Nrf2 stability and exacerbating its aberrant activation. Additionally, we demonstrated that ITGA6 enhances γ-secretase-mediated Notch signaling activation, ultimately promoting RBPJ-induced TRIM25 transcription. The current study provides the evidence supporting the effect of m6A modified HOXA10-AS and its downstream miR-29 b-3p/ITGA6 axis on regulating oxidative resistance and malignant progression in LSCC through the Notch and Keap1/Nrf2 pathways, and proposed that targeting this axis holds promise as a potential therapeutic approach for treating LSCC.