Laryngeal Squamous Cell Carcinoma Tissues Research Articles

Calreticulin-Driven Autophagy Enhances Cell Proliferation in laryngeal Squamous Cell Carcinoma

BackgroundCalreticulin (CALR) is a multifunctional calcium-binding protein. Recent studies have revealed that CALR contributes to tumor development and promotes cancer cell proliferation. However, how CALR affects the development of laryngeal squamous cell carcinoma (LSCC) remains mysterious. Thus, this study aimed to explore the effect of CALR on LSCC development and uncover its underlying mechanisms. MethodsCALR expression in LSCC cell lines and tissues was examined by qRT-PCR and western blot analysis and its functional role was detected via in vivo and in vitro assays. Cell proliferation was discriminated with CCK-8 and colony formation assays, while apoptosis was analyzed using flow cytometry. Autophagy levels were measured via LC3 immunofluorescence, and western blot assay was conducted to assess apoptosis- and autophagy-related proteins. Additionally, a mouse xenograft model was employed to determine the impact of CALR knockdown on tumor growth. ResultsWe found that CALR knockdown reduced LSCC cell viability and proliferation while enhancing apoptosis, whereas CALR overexpression showed opposite effects. In vivo experiments verified that CALR knockdown suppressed tumor growth. In addition, elevated CALR expression induced autophagy in LSCC cells, while autophagy inhibitor 3-MA (2.5mM) reversed the anti-apoptosis effects of CALR overexpression. ConclusionOur study identifies CALR as an oncogene in LSCC, where it promotes tumor progression by inducing autophagy and inhibiting apoptosis. Targeting CALR or modulating autophagy may represent novel therapeutic strategies for LSCC.

Hsa_circYARS interacts with miR-29a-3p to up-regulate IREB2 and promote laryngeal squamous cell carcinoma progression

ObjectiveThis study was to investigate the carcinogenic capacity of circYARS in laryngeal squamous cell carcinoma (LSCC) and to reveal its potential mechanism as a competitive endogenous RNA.MethodsThe differentially expressed circRNA and mRNA in LSCC were detected by RT-qPCR. Dual luciferase reporter assay and RIP were conducted to test the interaction between circYARS, miR-29a-3p, and IREB2. The functional effects of these molecules were investigated by CCK-8, flow cytometry, colony formation assay, Transwell, Western blot, and xenotransplantation mouse models.ResultsIn LSCC tissues and cell lines, circYARS and IREB2 levels were enhanced, while miR-29a-3p level was lowered. Depleting circYARS led to decreased IREB2 by promoting miR-29a-3p expression. As a result of miR-29a-3p enhancement or circYARS silence, the proliferative, migratory, and invasion of cancer cells were suppressed and apoptosis was stimulated.ConclusioncircYARS is involved in the tumorigenicity and progression of LSCC through the miR-29a-3p/IREB2 axis, providing strategies and targets for therapeutic intervention of LSCC.

Hsa_circ_0000825 promotes the progression of laryngeal squamous cell carcinoma by sponging miR-766 and interacting with ELAVL1

Emerging evidence suggests that circular RNAs (circRNAs) are involved in the regulation of tumourigenesis and progression of a variety of malignant tumours. In this study, we aimed to identify laryngeal squamous cell carcinoma (LSCC)-specific circRNAs and explore their biological functions and underlying molecular mechanisms. Employing microarray and qRT-PCR, hsa_circ_0000825 was found to be significantly increased in LSCC tissues versus para-cancerous tissues. High hsa_circ_0000825 expression was positively associated with advanced clinical stages, lymph node metastasis, and poor survival. Furthermore, the overexpression of hsa_circ_0000825 in TU177 and AMC-HN-8 cells promoted cell proliferation. Transwell assays showed enhanced migration and invasion of TU177 and AMC-HN-8 cells upon overexpression of hsa_circ_0000825. Conversely, the knockdown of hsa_circ_0000825 had the opposite effect. Xenograft tumours in BALB/c nude mice derived from hsa_circ_0000825-overexpressed TU177 cells showed greater volume and weight than those derived from control TU177 cells. Mechanistically, nuclear-cytoplasmic fractionation assay confirmed that hsa_circ_0000825 was mainly located in the cytoplasm of TU177 and AMC-HN-8 cells. The AGO2-RNA immunoprecipitation (RIP) assay revealed that hsa_circ_0000825 was significantly enriched in the AGO2-precipitated complex in both TU177 and AMC-HN-8 cells, suggesting that this circRNA may function via a competitive endogenous RNA (ceRNA) mechanism. Next, bioinformatics analysis, biotinylated-oligo pull-down assay and dual-luciferase reporter assay verified that miR-766 could be sponged by hsa_circ_0000825 and also target 3′UTR of HOXD10 mRNA. Moreover, miR-766 was shown to be involved in the pro-oncogenic effect of hsa_circ_0000825. This occurred via the mediation of hsa_circ_0000825-enhanced HOXD10 mRNA by the ceRNA mechanism in TU177 and AMC-HN-8 cells. Besides, RNA-binding protein (RBP) ELAVL1 interacted with hsa_circ_0000825 in TU177 and AMC-HN-8 cells, as revealed through bioinformatics analysis, biotinylated-oligo pull-down assays, and RIP assays. ELAVL1 knockdown decreased cell proliferation by 38 % and 34 % in hsa_circ_0000825-overexpressed TU177 and AMC-HN-8 cells (P < 0.05). Similarly, ELAVL1 was involved in the pro-migration and pro-invasion effects of hsa_circ_0000825 overexpression. In addition, comprehensive analysis of mRNA-seq in hsa_circ_0000825-overexpressed TU177 cells, as well as catRAPID and TCGA databases, suggested that ITGB2, HOXD10, and MTCL1 might be crucial downstream target mRNAs of ELAVL1 in LSCC, participating in the hsa_circ_0000825-ELAVL1 axis pro-oncogenic effect. Taken together, hsa_circ_0000825 plays a pro-oncogenic role in LSCC via the miR-766/HOXD10 axis and ELAVL1 and may serve as a promising specific biomarker and therapeutic target for LSCC.

Unlocking the potential of HHLA2: identifying functional immune infiltrating cells in the tumor microenvironment and predicting clinical outcomes in laryngeal squamous cell carcinoma

BackgroundHHLA2 (human endogenous retrovirus-H long terminal repeat-associating protein 2) represents a recently identified member of the B7 immune checkpoint family, characterized by limited expression in normal tissues but notable overexpression in various cancer types. Nevertheless, the precise function and interaction with immune cells remain poorly understood, particularly in laryngeal squamous cell carcinoma (LSCC). This investigation endeavored to elucidate the biological significance of HHLA2 within the tumor microenvironment of human LSCC tissues and delineate the clinical relevance and functional roles of HHLA2 in LSCC pathogenesis.MethodsThrough multiplexed immunohistochemistry analyses conducted on tissue microarrays sourced from LSCC patients (n = 72), the analysis was executed to assess the expression levels of HHLA2, density and spatial patterns of CD68+HLA-DR+CD163− (M1 macrophages), CTLA-4+CD4+FoxP3+ (CTLA-4+Treg cells), CTLA-4+CD4+FoxP3− (CTLA-4+Tcon cells), exhausted CD8+T cells, and terminally exhausted CD8+T cells in LSCC tissues. Survival analysis was conducted to evaluate the prognostic significance of HHLA2 and these immune checkpoints or immune cell populations, employing COX regression analysis to identify independent prognostic factors.ResultsKaplan–Meier (K–M) survival curves revealed a significant association between HHLA2 expression and overall survival (OS) in LSCC. Elevated levels of HHLA2 were linked to reduced patient survival, indicating its potential as a prognostic marker (HR: 3.230, 95%CI 0.9205–11.34, P = 0.0067). Notably, increased infiltration of CD68+ cells (total macrophages), STING+CD68+HLA-DR+CD163− (STING+M1 macrophages), CTLA-4+CD4+FoxP3+, CTLA-4+CD4+FoxP3−, PD-1+LAG-3+CD8+T cells, and PD-1+LAG-3+TIM-3+CD8+T cells strongly linked to poorer survival outcomes (P < 0.05). A discernible trend was observed between the levels of these immune cell populations, STING+CD68+ (STING+ total macrophages), CD68+HLA-DR+CD163−, STING+CD68+CD163+HLA-DR− (STING+M2 macrophages), PD-1+LAG-3−CD8+T cells, PD-1+TIM-3+CD8+T cells, and PD-1+LAG-3+TIM-3−CD8+T cells and prognosis. Importantly, multivariate COX analysis identified HHLA2 as an independent predictive factor for OS in LSCC patients (HR = 3.86, 95%CI 1.08–13.80, P = 0.038). This underscored the potential of HHLA2 as a critical marker for predicting patient outcomes in LSCC.ConclusionsHHLA2 emerged as a detrimental prognostic biomarker for assessing OS in LSCC patients. Relative to other immune checkpoints, HHLA2 exhibited heightened predictive efficacy for the prognosis of LSCC patients.

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.

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.

CircRNA hsa_circ_0069,399 as a potential clinical prognostic marker in laryngeal squamous cell carcinoma

ObjectiveCircular RNAs (circRNAs) significantly influence the invasion, metastasis, gene expression, proliferation, and apoptosis of tumor cells. However, the roles of circRNAs in laryngeal squamous cell carcinoma (LSCC) remain largely unexplored. This study aims to examine circRNA expression patterns in LSCC and adjacent non-tumorous tissues, with the goal of uncovering potential biomarkers for LSCC. MethodsTissue samples were collected from both the tumor and adjacent normal tissues of ten patients who had undergone surgical resection. The profiling of circRNAs was conducted through transcriptomic sequencing and analytical bioinformatics approaches. A ternary regulatory network based on the competitive endogenous RNA (ceRNA) hypothesis was established, linking target circRNAs to clinical immunohistochemical parameters for comparison. Verification of target circRNAs in LSCC tissues was performed using quantitative real-time PCR (RT-qPCR), whereas target mRNAs were analyzed through immunohistochemistry. ResultsA total of 126 significantly different circRNAs were identified, including 40 up-regulated genes and 86 down-regulated genes. Furthermore, 92 circRNA-miRNA-mRNA regulatory relationship axes related to clinical immunohistochemical indicators were found based on 5 candidate circRNAs. Interestingly, all axes related to the target genes MKI67 and TP53 were found to compete with the same circRNA: hsa_circ_0069,399. Further verification confirmed that the hsa_circ_0069,399 expression was overtly upregulated in tumor tissues from LSCC patients, which was consistent with the sequencing results. Conclusionhsa_circ_0069,399 could be a potential prognostic marker for LSCC.

LSM6 promotes cell proliferation and migration regulated by HMGB1 in laryngeal squamous cell carcinoma

Elevated levels of high mobility group protein B1 (HMGB1) play a significant role in the pathogenesis of many diseases, but is particularly important for the formation of malignant tumors. Nonetheless, the function of HMGB1 and the underlying mechanism of laryngeal squamous cell carcinoma (LSCC) remain incompletely understood, causing uncertainty. Here we found immunohistochemistry from 97 LSCC tissues showed HMGB1 was upregulated, which was associated with poor differentiation. HMGB1 knockdown could significantly inhibit wound closure and colony formation. The full-genome gene expression microarray was performed to investigate the mechanism. After knockdown of HMGB1 by siRNA, among the expressed differential genes, 10 genes were randomly selected for validation. Then, shRNA lentivirus targeting these genes were constructed to explore their role in LSCC by cell proliferation assay. LSM6 downregulation was dramatically promoted by HMGB1 knockdown, resulting in higher expression in LSCC tissues. Furthermore, downregulation of LSM6 could significantly suppress cell proliferation, migration and colony formation. This study indicated that HMGB1 promoted LSCC cell malignant phenotypes through regulation of LSM6. We anticipate that HMGB1-LSM6 could be a putative therapeutic target for LSCC.

FAM83B promotes cell proliferation via regulating the expression of CDK4/CDK6/CCND1 complex in laryngeal squamous cell carcinoma

FAM83B, as one of the FAM83 family members, has been closely involved in cell transformation, and a growing number of scholars have been studied its role in tumours over the years. Whereas the effect and potential mechanism of FAM83B in laryngeal squamous cell carcinoma (LSCC) have not been investigated. In this research, we discovered that the expression quantity of FAM83B was remarkably higher in LSCC tissues (79.65 ± 35.98) than in matched adjacent tissues (59.34 ± 32.59) by tissue microarrays and immunohistochemistry. Furthermore, expression of FAM83B was knocked down in HEP-2 and TU177 cell lines via lentivirus, and in the course of intracorporal and extracorporeal experiments, FAM83B knockdown showed the inhibition of tumour growth, migration, and invasion ability. Moreover, cell cycle assay showed that FAM83B knockdown leads to an apparent accumulation of cells in the G1 phase, indicating that FAM83B knockdown can inhibit cell proliferation. Meanwhile, western blotting (WB) demonstrated that FAM83B knockdown led to a significant reduction in CDK4/CDK6/CCND1 protein expression, which may have decelerated cell cycle progression. Collectively, this study demonstrates that FAM83B serves as an oncogene in LSCC, promoting cell proliferation by controlling the protein expression of CDK4, CDK6, and CCND1, thus inducing a transference of the G1 stage to S stage in cell-cycle of LSCC cells. These results provide an academic foundation for elucidating the mechanism of LSCC occurrence and evolution and for developing treatment strategies for LSCC.

Curcumin suppresses the malignant phenotype of laryngeal squamous cell carcinoma through downregulating E2F1 to inhibit FLNA.

Curcumin is a kind of polyphenol substance extracted from the rhizome of Curcuma longa. Because of its good biological activity and pharmacological effects, it has been used in anti-tumor research. The aim of this study was to investigate the anti-cancer mechanism of curcumin on laryngeal squamous cell carcinoma (LSCC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to check the expression level of transcription factor E2F1 (E2F1) and filamin A (FLNA) mRNA. E2F1 and FLNA protein and proliferation-associated protein were detected through western blot. Cell viability was showed by MTT assay, and flow cytometry was used to exhibit cell cycle distribution and cell apoptosis. Tube formation assay was used to detect the angiogenesis ability of cells. Transwell was used as a method to observe cell migration and invasion. The online website JASPAR predicted the binding site of E2F1 and FLNA promoter, and chromatin immunoprecipitation (ChIP) and dual-luciferase report experiment verified the combination. Curcumin treatment made LSCC cells viability reduce, cell cycle retardant, angiogenesis decrease, metastasis inhibition and apoptosis increase. And curcumin treatment could downregulate the expression of E2F1, and E2F1 overexpression would reverse the influence of curcumin treatment in LSCC cells. Moreover, E2F1 could bind to FLAN promoter and promote FLNA expression. The expression level of FLNA was higher in LSCC tissue and cells compared with normal tissue and cells. E2F1 knockdown inhibited malignant phenotype of LSCC cells, which would be reversed by FLNA addition. In addition, FLNA had high level in LSCC tissue and cells. Curcumin regulated FLNA expression via inhibiting E2F1. Finally, in vivo assay showed that curcumin inhibition restrained LSCC tumor formation. Curcumin downregulated FLNA expression through inhibiting E2F1, thereby suppressing the malignant phenotype and angiogenesis of LSCC cells, which was a new regulatory pathway in LSCC.

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

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. 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. 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. 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.

IL-17 expression and prognosis of laryngeal squamous cell carcinoma.

Objective: To investigate the roles of proinflammatory cytokine IL-17 in the prognosis of laryngeal squamous cell carcinoma (LSCC). Methods: In all, 67 LSCC patients were enrolled to evaluate the expression of IL-17, IL-23, CD8, CD31, and vascular endothelial growth factor by immunohistochemical staining. Cumulative survival time was determined by the Kaplan-Meier approach, and prognostic variables for overall survival (OS) were identified by Cox proportional-hazards models. Results: The expression of IL-17 and IL-23 was remarkably higher in LSCC tissue than in adjacent normal (AN) tissue (P < .001). The Kaplan-Meier analysis indicated that patients with cervical lymph node metastasis (CLNM), advanced tumor node metastasis (TNM) stage, poor-differentiated grade, high microvessel density (MVD) in LSCC tissues, and high IL-17 expression in AN tissues had poorer OS (all P < .05). Cox regression analysis revealed that CLNM (HR = 7.246; 95% CI: 2.598-20.208; P < .001), high MVD in tumor tissues (HR = 4.233; 95% CI: 1.482-12.092; P = .007), and low CD8 expression in AN tissues (HR = 3.996; 95% CI: 1.442-11.074; P = .008) were significantly associated with poorer OS in LSCC, while IL-17 expression in AN tissue was not. Conclusion: High IL-17 expression in AN tissues could indicate poor prognosis and may be a prognostic marker for LSCC. Low CD8 expression in AN tissues may represent poor frontline antitumor immunity and be associated with poor prognosis.

IGF2BP2-m6A-circMMP9 axis recruits ETS1 to promote TRIM59 transcription in laryngeal squamous cell carcinoma

Laryngeal squamous cell carcinoma (LSCC) is a common malignancy of the head and neck. Recently, circular RNA (circRNA) has been studied extensively in multisystem diseases. However, there are few research on biological functions and molecular mechanisms of circRNAs in LSCC. CircRNA array was used to detect the differentially expressed circRNAs. Kaplan–Meier and cox regression analysis were used to identify survival based on circMMP9. The qRT-PCR, RNase R treatment, sanger sequencing and in situ hybridization were used to verify circMMP9 expression, characteristics and localization in LSCC tissues and cells. Functionally, colony formation, MTS, transwell and in vivo assays were proceeded to detect the biological function of circMMP9 in LSCC progression. The RNA-seq was conducted to identify the molecular targets of circMMP9. Mechanically, MeRIP, RNA Immunoprecipitation (RIP), RNA pulldown, Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were carried on to verify the regulatory mechanism of circMMP9. CircMMP9 was discovered upregulated in LSCC tissues and cells, and high level of circMMP9 was associated with poor prognosis, low degree of pathological grading, high TNM stage and lymph node metastasis of LSCC. CircMMP9 knockdown prevented LSCC progression both in vitro and in vivo, whereas, circMMP9 overexpression had the opposite effect. CircMMP9 was stabilized by IGF2BP2 in m6A-dependent manner. TRIM59 was identified as downstream target of circMMP9. CircMMP9 recruited ETS1 to stimulate TRIM59 transcription. Moreover, TRIM59 accelerated LSCC progression via activating the PI3K/AKT signal pathway. Our findings offered a unique regulatory mechanism for circMMP9 in LSCC, as well as a novel proof that circMMP9 may be utilize as a diagnostic marker and therapeutic target for LSCC patients.

Exosomal circMACF1 drives PI3K/AKT/mTOR-mediated autophagy suppression in laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor. The regulatory functions of circular RNAs (circRNAs) in cancers have been broadly reported. The hsa_circ_0011773 (circMACF1) is reported to be overexpressed in LSCC tissues, while its biological function in LSCC remains unclear. CircMACF1 expression in LSCC tissues and cells was assessed via RT-qPCR. Exosomes extracted from cells were identified by TEM and NTA. Autophagy-related proteins were tested by western blot. Confocal microscope was employed for analyzing LC3 expression. Cell proliferation, migration, and invasion were assessed by CCK-8 assay and transwell assay. The levels of main proteins on PI3K/AKT/mTOR were tested by western blot. We observed that circMACF1 was highly expressed in LSCC tissues and cells. Furthermore, circMACF1 expression was also upregulated in the exosomes derived from LSCC cells. CircMACF1 depletion promoted LC3 expression in cells. Additionally, we proved that circMACF1 knockdown suppressed LSCC cell proliferative, migratory and invasive capabilities via promoting autophagy. Exosomal circMACF1 was found to promote LSCC tumor growth. Then, we proved that circMACF1 could activate PI3K/AKT/mTOR pathway to regulate autophagy. Moreover, MACF1 was positively regulated by circMACF1 and its overexpression notably reversed the effects of circMACF1 depletion in LSCC progression. Exosomal circMACF1 can regulate PI3K/AKT/mTOR-mediated autophagy suppression to facilitate LSCC development.

Interleukin-4 mRNA Expression in Laryngeal Squamous Cell Carcinoma Patients with or without Lymph Node Involvement

Background: Interleukin (IL)-4 is a member of T helper 2 (Th2) axis produced by T-lymphocyte and mast cell. It has been shown that IL-4 expression changes in tumor tissues. The main objective of this study is to investigate the expression of IL-4 mRNA in patients with Laryngeal Squamous Cell Carcinoma (LSCC) with or without lymph node involvement. Materials and Methods: mRNA expression of IL-4 in LSCC tissues were detected by quantative Real-Time PCR (qRT-PCR). Expression of IL-4 gene was compared between lymph node positive and negative patients with Squamous Cell Carcinoma of Larynx. Results: No statistically significant association was found in expression of IL-4 between lymph node positive and negative patients. Conclusion: It seems that IL-4 has no important effect on the involvement of lymph node in LSCC. However, to achieve a definite conclusion more investigations are certainly required.

Triosephosphate isomerase 1 may be a risk predictor in laryngeal squamous cell carcinoma: a multi-centered study integrating bulk RNA, single-cell RNA, and protein immunohistochemistry

BackgroundAlthough great progress has been made in anti-cancer therapy, the prognosis of laryngeal squamous cell carcinoma (LSCC) patients remains unsatisfied. Quantities of studies demonstrate that glycolytic reprograming is essential for the progression of cancers, where triosephosphate isomerase 1 (TPI1) serves as a catalytic enzyme. However, the clinicopathological significance and potential biological functions of TPI1 underlying LSCC remains obscure.MethodsWe collected in-house 82 LSCC tissue specimens and 56 non-tumor tissue specimens. Tissue microarrays (TMA) and immunohistochemical (IHC) experiments were performed. External LSCC microarrays and bulk RNA sequencing data were integrated to evaluate the expression of TPI1. We used a log-rank test and the CIBERSORT algorithm to assess the prognostic value of TPI1 and its association with the LSCC microenvironment. Malignant laryngeal epithelial cells and immune-stromal cells were identified using inferCNV and CellTypist. We conducted a comprehensive analysis to elucidate the molecular functions of TPI1 in LSCC tissue and single cells using Pearson correlation analysis, high dimensional weighted gene co-expression analysis, gene set enrichment analysis, and clustered regularly interspaced short palindromic repeats (CRISPR) screen. We explored intercellular communication patterns between LSCC single cells and immune-stromal cells and predicted several therapeutic agents targeting TPI1.ResultsBased on the in-house TMA and IHC analysis, TPI1 protein was found to have a strong positive expression in the nucleus of LSCC cells but only weakly positive activity in the cytoplasm of normal laryngeal cells (p < 0.0001). Further confirmation of elevated TPI1 mRNA expression was obtained from external datasets, comparing 251 LSCC tissue samples to 136 non-LSCC tissue samples (standardized mean difference = 1.06). The upregulated TPI1 mRNA demonstrated a high discriminative ability between LSCC and non-LSCC tissue (area under the curve = 0.91; sensitivity = 0.87; specificity = 0.79), suggesting its potential as a predictive marker for poor prognosis (p = 0.037). Lower infiltration abundance was found for plasma cells, naïve B cells, monocytes, and neutrophils in TPI-high expression LSCC tissue. Glycolysis and cell cycle were significantly enriched pathways for both LSCC tissue and single cells, where heat shock protein family B member 1, TPI1, and enolase 1 occupied a central position. Four outgoing communication patterns and two incoming communication patterns were identified from the intercellular communication networks. TPI1 was predicted as an oncogene in LSCC, with CRISPR scores less than -1 across 71.43% of the LSCC cell lines. TPI1 was positively correlated with the half maximal inhibitory concentration of gemcitabine and cladribine.ConclusionsTPI1 is dramatically overexpressed in LSCC than in normal tissue, and the high expression of TPI1 may promote LSCC deterioration through its metabolic and non-metabolic functions. This study contributes to advancing our knowledge of LSCC pathogenesis and may have implications for the development of targeted therapies in the future.

M6A reader YTHDF1 promotes malignant progression of laryngeal squamous carcinoma through activating the EMT pathway by EIF4A3

Laryngeal squamous cell carcinoma (LSCC) is one of the common malignant tumors in the head and neck region, and its high migration and invasion seriously threaten the survival and health of patients. In cancer development, m6A RNA modification plays a crucial role in regulating gene expression and signaling. This study delved into the function and mechanism of the m6A reading protein YTHDF1 in LSCC. It was found that YTHDF1 was highly expressed in the GEO database and LSCC tissues. Cell function experiments confirmed that the downregulation of YTHDF1 significantly inhibited the proliferation, migration, and invasion ability of LSCC cells. Further studies revealed that EIF4A3 was a downstream target gene of YTHDF1, and knockdown of EIF4A3 similarly significantly inhibited the malignant progression of LSCC in both in vivo and in vitro experiments. The molecular mechanism studies suggested that YTHDF1-EIF4A3 may promote the malignant development of LSCC by activating the EMT signaling pathway. This study provides important clues for an in-depth understanding of the pathogenesis of LSCC and is a solid foundation for the discovery of new therapeutic targets and approaches.

Upregulated TC1 and downregulated Chibby were correlated with the aberrant β-catenin expression in laryngeal squamous cell carcinoma.

As an important member of Wnt/β-catenin signaling pathway, the aberrant expression of β-catenin has been implicated in many cancers. Chibby, a β-catenin binding partner, is an antagonist involved in this pathway. In contrast, thyroid cancer 1 (TC1) as an activator of this pathway can relieve the antagonistic activity of Chibby on the β-catenin-mediated transcription and is high expressed in human tumors. The objectives of this study were to examine the expression of TC1, Chibby, and β-catenin and investigate the association among them in laryngeal squamous cell carcinoma (LSCC). The expression of TC1, Chibby, β-catenin, c-Myc, Cyclin D1, and matrix metalloproteinase-7 (MMP-7) were examined by immunohistochemistry in samples from 53 LSCC patients. Compared with normal laryngeal squamous epithelium (NLSE), there were upregulated expression of TC1, downregulated expression of Chibby, and aberrant cytoplasmic expression of β-catenin in the LSCC tissues (P < .001). The high expression of TC1 was correlated with the tumor site, advanced TNM and T stage, lymphovascular invasion, and poor differentiation in LSCC tissues (P < .050). There were correlations between the aberrant expression of β-catenin and the tumor site, advanced TNM and T stage, lymphovascular invasion, perineurial invasion, and poor differentiation in LSCC tissues (P < .050). Upregulated TC1 and downregulated Chibby were correlated with aberrant expression of β-catenin (P < .001), but no correlation between them (P = .076). The percent of abnormal expression of β-catenin in LSCC was 96.00% in TC1+/Chibby-, 73.68% in TC1+/Chibby+, 0.00% in TC1-/Chibby-, and 0.00% in TC1-/Chibby + group (P < .001). High expression of c-Myc, Cyclin D1, and MMP-7 was observed in LSCC tissues (P < .001). There was statistically significant about the expression of Cyclin D1 and MMP-7 among the groups of TC1+/Chibby-, TC1+/Chibby+, TC1-/Chibby-, and TC1-/Chibby + (P < .001), but was not significance about the expression of c-Myc among them (P = .339). No association was found between overall survival and the expression of TC1, Chibby, and β-catenin (P > .05). The upregulated expression of TC1 and downregulated expression of Chibby were correlated with the aberrant expression of β-catenin and the high expression of Cyclin D1 and MMP-7 in LSCC tissues.

NAT10 promotes the tumorigenesis and progression of laryngeal squamous cell carcinoma through ac4C modification of FOXM1 mRNA

ABSTRACT Laryngeal squamous cell carcinoma (LSCC), is a prevalent malignant tumor, belongs to the category of head and neck tumors. N-acetyltransferase 10 (NAT10) can alter mRNA stability through N4- acetylcytidine (ac4C) modification. This study aimed to make an investigation into the role of NAT10-mediated ac4C modification in the malignant processes of LSCC cells. The NAT10 expression in LSCC tissues and cells was detected RT-qPCR and western blot. The ac4C dot blot was performed to detect ac4C level. Besides, the cell viability, migration, and invasion abilities were detected by CCK-8 and transwell assays. AcRIP-qPCR was performed to measure the abundance of ac4C on FOXM1 mRNA. RIP and Luciferase reporter assays were performed to demonstrate the interaction between NAT10 and FOXM1. Finally, the xenograft model was established to explore the role of NAT10 in vivo. NAT1 levels were significantly increased in the LSCC tissues and cells. Knockdown of NAT10 could significantly suppress the proliferation, migration, and invasion of LSCC cells. Additionally, NAT10 recognized the ac4C-modified sites in the 3’-untranslated regions (3’ UTR) of forkhead box M1 (FOXM1) to enhance the ability of FOXM1 mRNA. Furthermore, FOXM1 overexpression reversed the suppressing effects of NAT10 knockdown on the proliferation, migration, and invasion of LSCC cells, according to the results of rescue assays. Finally, results of animal experiments showed that NAT10 promoted in vivo tumorigenesis of LSCC cells through upregulating FOXM1. Our current study demonstrated that NAT10-mediated ac4C modification of FOXM1 mRNA promoted the malignant processes of LSCC cells.

Exosomal lncRNA LINC02191 Promotes Laryngeal Squamous cell Carcinoma Progression by Targeting miR-204-5p/RAB22A Axis and Regulating PI3K/Akt/mTOR Pathway.

Recent research has explored the potential use of serum-derived biomarkers in cancer screening, and mounting evidence has illustrated the pivotal roles of long noncoding RNAs (lncRNAs) in regulating laryngeal squamous cell carcinoma (LSCC) progression. LINC02191 is a newly identified lncRNA and no studies have investigated its role in malignant tumors. This study aims to explore the functions and mechanisms of lncRNA LINC02191 in LSCC. LINC02191 was knocked down in LSCC cells using shRNAs for loss-of-function experiments. RT-qPCR revealed that LINC02191was upregulated in LSCC patients' serum exosomes, tissues and cells. Western blotting and RT-qPCR were implemented for detecting molecular protein and RNA levels. Colony formation, CCK-8, wound healing and Transwell assays were employed for examining LSCC cell malignant behaviors in vitro. A tumor-bearing mouse model (n = 4/group) was established for examining LINC02191 role in vivo.The results showed that LINC02191 silencing hindered LSCC cell proliferation, invasiveness, migration as well as EMT in vitro and impeded tumorigenesis in xenograft mouse model. Luciferase reporter assay was utilized for verifying the interaction between LINC02191, miR-204-5p and RAB22A. Pearson correlation analysis was employed to evaluate their expression correlation in LSCC tissue specimens (N = 30). Mechanistically, LINC02191 upregulated RAB22A by binding to miR-204-5p, and knocking down LINC02191 inhibited PI3K/Akt/mTOR signaling transduction in LSCC cells and tumor-bearing mice. Moreover, RAB22A overexpression reversed LINC02191 depletion-triggered suppression of LSCC cell aggressiveness and inactivation of PI3K/Akt/mTOR signaling. In conclusion,LINC02191 aggravates LSCC by targeting miR-204-5p/RAB22A/PI3K/Akt/mTOR signaling pathway, which indicates that LINC02191 may serve as a promising target for LSCC treatment.