Expression In Laryngeal Squamous Cell Carcinoma Research Articles

Integrative methylome and transcriptome analysis reveals epigenetic regulation of Fusobacterium nucleatum in laryngeal cancer.

The aetiological mechanisms of Fusobacterium nucleatum in laryngeal cancer remain unclear. This study aimed to reveal the epigenetic signature induced by F. nucleatum in laryngeal squamous cell carcinoma (LSCC). Combined analysis of methylome and transcriptome data was performed to address the functional role of F. nucleatum in laryngeal cancer. Twenty-nine differentially expressed methylation-driven genes were identified by mapping the methylation levels of significant differential methylation sites to the expression levels of related genes. The combined analysis revealed that F. nucleatum promoted Janus kinase 3 (JAK3) gene expression in LSCC. Further validation found decreased methylation and elevated expression of JAK3 in the F. nucleatum-treated LSCC cell group; F. nucleatum abundance and JAK3 gene expression had a positive correlation in tumour tissues. This analysis provides a novel understanding of the impact of F. nucleatum in the methylome and transcriptome of laryngeal cancer. Identification of these epigenetic regulatory mechanisms opens up new avenues for mechanistic studies to explore novel therapeutic strategies.

MiR-125a-5p regulates the radiosensitivity of laryngeal squamous cell carcinoma via HK2 targeting through the DDR pathway.

To determine the function of miR-125a-5p in laryngeal squamous cell carcinoma (LSCC), its correlation with radiation sensitivity, and the underlying regulatory mechanism. We conducted the analysis on the correlation between miR-125a-5p and head and neck squamous cell carcinoma (HNSCC) using data obtained from The Cancer Genome Atlas (TCGA). The putative gene targeted by miR-125a-5p has been identified as HK2, while the expression levels of miR-125a-5p and HK2 were measured in laryngeal cancer tissues and cells using RT-PCR. MiR-125a-5p and HK2 were introduced into the lentiviral vector and the vector was used to transfect AMC-HN-8 cells. The roles of miR-125a-5p and HK2 in LSCC and on radiosensitivity were determined by evaluating cell growth, examining colony formation, analyzing flow cytometry, and utilizing the single hit multi-target model. Western blotting was used to measure H2AX and rH2AX levels in the DNA damage response (DDR) pathway. The validation of the interaction between miR-125a-5p and HK2 was conducted through the dual-luciferase assay. To further confirm the association between miR-125a-5p and HK2, as well as its influence on radiosensitivity, rescue experiments were performed. The expression of miR-125a-5p is downregulated in LSCC, while upregulating its expression could suppress cell growth, induce apoptosis, and enhance radiosensitivity. Additionally, HK2 exhibited high expression in LSCC and the biological function was opposite to miR-125a-5p. Western blotting analysis revealed that miR-125a-5p increased rH2AX levels and decreased H2AX levels, conversely, HK2 had the opposite effect on miR-125a-5p. These findings suggested that HK2 may serve as the target gene of miR-125a-5p. The double luciferase assay confirmed the binding of HK2 to miR-125a-5p, and rescue trials confirmed the role of miR-125a-5p in regulating the effects and radiation sensitivity of LSCC by targeting HK2 via the DDR pathway. By targeting HK2 and impacting the DDR pathway, miR-125a-5p has been found to inhibit cellular proliferation, enhance apoptosis, and heighten radiosensitivity in LSCC.

Integrative Analysis Reveals STC2 as a Prognostic Biomarker of Laryngeal Squamous Cell Carcinoma.

Stanniocalcin 2 (STC2) is involved in many tumour types, but it remains unclear what its biological function is in laryngeal squamous cell carcinoma (LSCC). Therefore, we investigated STC2's expression, potential function, and prognostic significance of in LSCC. The expression and prognosis of STC2 in LSCC were described using the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. In the TCGA database, the relationship between STC2 and immune infiltration, expression of immune cell chemokine and receptor genes, immune cell molecular marker genes, and epithelial‒mesenchymal transition (EMT) marker genes were analysed. The biological processes involved in STC2 and its expression-related genes were analysed comprehensively using bioinformatics. The single-gene ceRNA network of STC2 was constructed in the TCGA database. Finally, LSCC patients' tumour tissue STC2 expression was verified. STC2 silencing with the RNAi technique was used for the determination of cellular functions in a laryngeal cancer cell line. STC2 expression was higher in most tumours, including LSCC, than in normal tissues and was associated with poor prognosis. The relative proportions of naïve B, plasma, follicular helper T, and macrophage M0 cells in LSCC and normal samples differed significantly. STC2 expression correlated significantly positively with that of TGFB1 (biomarker of Tregs) and significantly negatively with that of D79A and CD19 (biomarkers of B cells). Furthermore, STC2 affected chemokine and receptor gene expression in immune cells. STC2 expression correlated with EMT marker gene expression in LSCC. STC2 was enriched in the PI3K/AKT signalling pathway, extracellular matrix (ECM) organisation, ECM-receptor interaction, and other tumour-related signalling pathways. STC2 was highly expressed in our clinical samples. N-cadherin and vimentin expression were decreased in the TU686 cell line after successful silencing of STC2, indicating that high STC2 expression may prompt LSCC cells to adopt a mesenchymal cell phenotype. STC2 silencing substantially reduced proliferation and migration in the TU686 cell line. STC2 may be a promising predictive biomarker for tumours, providing new approaches for LSCC diagnosis and treatment monitoring.

PTPN12 down-regulated by miR-146b-3p gene affects the malignant progression of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is a common malignancy among men in the anatomical position of head and neck. Hoarseness, pharyngalgia, and dyspnea are common symptoms. LSCC is a complex polygenic carcinoma that is caused by many factors involving polygenic alteration, environmental pollution, tobacco, and human papillomavirus. Classical protein tyrosine phosphatase nonreceptor type 12 (PTPN12) has been extensively studied to decipher its mechanism as a tumor suppressor gene in various human carcinomas; however, there is no comprehensive elucidation of the PTPN12 expression and its regulatory mechanisms in LSCC. As such, we expect to provide new insights for finding new biomarkers and effective therapeutic targets in LSCC. Immunohistochemical staining, western blot (WB), and quantitative real-time RT-PCR (qRT-PCR) were used for the messenger RNA (mRNA) and protein expression analyses of PTPN12, respectively. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, clone formation, transwell migration, and transwell invasion assays were used to assess the proliferation, migration, and invasion ability of LSCC cells. Online prediction and design software tools (http://www.targetscan.org/ and http://www.microRNA.org) were used to predict associated miRNA. Studying the targeted regulatory relationship between miR-146b-3p and PTPN12 was based on dual luciferase reporter gene analysis. qRT-PCR was used to assess miR-146b-3p expression in LSCC. miR-146b-3p inhibitor and mimic were transfected, followed by qRT-PCR and WB assays to detect the expression of PTPN12. The gain and loss functional experiments were used to investigate the effects of miR-146b-3p transfection on the proliferation, migration, and invasion of tumor cells. Online bioinformatics prediction software (https://cn.string-db.org/ and https://www.genecards.org/) was used to determine potential downstream target genes of PTPN12. qRT-PCR and WB analyses were used to assess the mRNA and protein expression levels of target genes. Our study showed significantly decreased mRNA and protein expression levels of PTPN12 in LSCC compared with the adjacent normal tissues. The lower PTPN12 mRNA expression was correlated with pathological differentiation, and lower PTPN12 protein expression was correlated with the TNM stage in LSCC tissues. The subsequent in vitro functional analyses showed the inhibitory effect of PTPN12 over-expression on the proliferation, migration, and invasiveness abilities of LSCC cell line. Using online prediction and design software, miR-146b-3p was searched to target PTPN12. The miR-146b-3p was expressed at a high level in LSCC tissues and cell lines. Luciferase reporter assay exhibited that miR-146b-3p inhibited the luciferase activity of PTPN12 markedly. The functional analyses showed the tumor-promoting role of miR-146b-3p on the proliferation, migration, and invasiveness abilities of LSCC cell. Furthermore, co-transfection of cells with miR-146b-3p and PTPN12 significantly restored the inhibitory effect of PTPN12 on LSCC cell growth, migration, and invasiveness. This phenomenon unveiled that miR-146b-3p regulated the proliferation, migration, and invasion of LSCC cells by targeting PTPN12. EGFR and ERBB2 were selected as the downstream-regulation target genes. Up-regulation of PTPN12 significantly suppressed EGFR expression. Accordingly, the miR-146b-3p mimic significantly up-regulated the EGFR expression. However, up-regulation of PTPN12 and miR-146b-3p mimic suppressed ERBB2 protein expression but induced its gene expression. Down-regulation of PTPN12 is associated with up-regulation of miR-146b-3p in LSCC. Moreover, PTPN12 serves as a tumor suppressor gene through regulating the proliferation, migration, and invasion of LSCC cells. miR-146b-3p/PTPN12 axis is expected to be a novel therapeutic target in LSCC.

Potentials of lncRNA-miRNA-mRNA networks as biomarkers for laryngeal squamous cell carcinoma.

Chemoresistance, radioresistance, and facile spreading of laryngeal squamous cell carcinoma (LSCC) make the practically clinical treatment invalid. Such dismal outcome mainly originates from the lack of effective biomarkers which are highly desirable to understand the pathogenesis of LSCC, and strives to find promising novel biomarkers to improve early screening, effective treatment, and prognosis evaluation in LSCC. Recently, long non-coding RNAs (lncRNAs), a kind of non-coding RNAs longer than 200 nucleotides, can participate in the process of tumorigenesis and progression through many regulatory modalities, such as epigenetic transcriptional regulation and post-transcriptional regulation. Meanwhile, microRNAs (miRNAs, miRs), essentially involved in the post-transcriptional regulation of gene expression, are aberrantly expressed in cancer-related genomic regions or susceptible sites. An increasing number of studies have shown that lncRNAs are important regulators of miRNAs expression in LSCC, and that miRNAs can also target to regulate the expression of lncRNAs, and they can target to regulate downstream messenger RNAs (mRNAs) transcriptionally or post-transcriptionally, thereby affecting various physiopathological processes of LSCC. Complex cross-regulatory networks existing among lncRNAs, miRNAs, and mRNAs can regulate the tumorigenesis and development of LSCC. Such networks may become promising biomarkers and potential therapeutic targets in the research field of LSCC. In this review, we mainly summarize the latest research progress on the regulatory relationships among lncRNAs, miRNAs, and downstream mRNAs, and highlight the potential applications of lncRNA-miRNA-mRNA regulatory networks as biomarkers for the early diagnosis, epithelial-mesenchymal transition (EMT) process, chemoresistance, radioresistance, and prognosis of LSCC, aiming to provide important clues for understanding the pathogenesis of LSCC and developing new diagnostic and therapeutic strategies.

Expression and clinical significance of FGFR1 and FGFR2 in laryngeal squamous cell carcinoma.

BackgroundFibroblast growth factor receptor 1 (FGFR1) and fibroblast growth factor receptor 2 (FGFR2) may be of significance in the development of laryngeal squamous cell carcinoma (SCC) tissues. Examination of the expression results of these factors may offer new insights into treatment of the disease, such as genetic and histological targeted target therapy.MethodsWe selected tissue from 30 cases of laryngeal SCC, 23 cases of adjacent normal mucosa, and 26 cases of benign laryngeal mucosal tissues from patients who received surgery at the Otolaryngology Department of the Affiliated Hospital of Chengde Medical College between September 2020 and January 2022. The laryngeal cancers included nine cases of supraglottic, 20 glottic (vocal cord), and one case of subglottic cancer, while all benign laryngeal mucosal lesions were obtained from vocal cord polyps. The expression of FGFR1 and FGFR2 was detected in 30 laryngeal cancers, 23 adjacent normal mucosa, and 26 vocal cord polyps by immunohistochemical technology [immunohistochemistry (IHC)], and the correlation analysis of their expression in laryngeal cancer was performed. P<0.05 was represented statistically significant.ResultsThe expression of FGFR1 and FGFR2 was significantly different in laryngeal SCC and the normal tissue >0.5 cm from the tumor margin (P<0.05), and between laryngeal SCC and vocal polyps (P<0.05). There was no difference in FGFR1 and FGFR2 expression (P>0.05) between normal mucosal margins and vocal cord polyp tissue, and no correlation between FGFR1 and FGFR2 in laryngeal SCC and sex, age, smoking history, alcohol consumption history, tumor diameter, tumor lymph node metastasis, tumor differentiation degree, and Tumor-Node-Metastasis (TNM) stage (P>0.05), A moderate positive correlation between FGFR1 expression and FGFR2 expression in laryngeal SCC was seen (Rs=0.499, P<0.01).ConclusionsFGFR1 and FGFR2 may participate in the occurrence of SCC of the throat: (I) positive FGFR1 and FGFR2 expressions are not associated with gender, age, smoking history, alcohol consumption history, tumor diameter, lymph node metastasis, degree of differentiation, or TNM stage. (II) FGFR2 increases successively with higher FGFR1 expression and with a positive correlation in laryngeal SCC.

Knockdown of circMYOF inhibits cell growth, metastasis, and glycolysis through miR-145-5p/OTX1 regulatory axis in laryngeal squamous cell carcinoma

New evidence suggests that abnormal expression of circular RNA (circRNA) is associated with the development of human cancers. This study aims to reveal circMYOF roles in the malignant phenotype of laryngeal squamous cell carcinoma (LSCC). The expression of circMYOF, microRNA (miR)-145-5p, and orthodenticle homeobox 1 (OTX1) was detected by quantitative real-time PCR. Cell proliferation, migration, invasion, and apoptosis were determined using colony formation assay and EdU assay, wound healing assay, transwell assay, and flow cytometry, respectively. Protein expression was examined by western blot analysis. Cell glycolysis was assessed by detecting glucose consumption and lactate production. Mice xenograft models were constructed to evaluate the regulation of circMYOF on LSCC tumorigenesis. The regulatory relationships among circMYOF, miR-145-5p, and OTX1 were identified using dual-luciferase reporter assay and RIP assay. Serum exosomes were isolated to confirm the existence of circMYOF in LSCC patients. CircMYOF was upregulated in LSCC tissues and cells, and its knockdown suppressed LSCC cell growth, metastasis, and glycolysis, as well as inhibited LSCC tumor growth. MiR-145-5p had decreased expression in LSCC, and it could be sponged by circMYOF. The inhibition effect of circMYOF lentivirus short hairpin RNA (sh-circMYOF) on LSCC progression was restored by the inhibitor of miR-145-5p (in-miR-145-5p). Also, OTX1 was targeted by miR-145-5p and was positively regulated by circMYOF. MiR-145-5p could repress LSCC progression, and OTX1 overexpression also eliminated this effect. In addition, we found that circMYOF was significantly overexpressed in the serum exosomes of LSCC patients. Our data revealed that circMYOF contributed to LSCC progression by promoting cell growth, metastasis, and glycolysis through miR-145-5p/OTX1 axis.

Ferroptosis in laryngeal squamous cell carcinoma and its regulation by M2 macrophage-derived exosomes

Objective: To investigate ferroptosis in laryngeal squamous cell carcinoma (LSCC) and its regulation by M2 macrophage-derived exosomes. Methods: LSCC and adjacent noncancerous tissue samples were collected from 32 patients treated in the Department of Otorhinolaryngology, Head and Neck Surgery of the Second Affiliated Hospital of Harbin between September 2018 and April 2021, including 26 males and 6 females, aged 43-79 years. The expressions of ferroptosis marker glutathione peroxidase 4(GPX4) in LSCC and adjacent noncancerous tissues were detected by immunohistochemistry and reverse transcriptase-polymerase chain reaction(RT-PCR). The correlations between GPX4 expression and clinicopathological factors in LSCC were analyzed. Biological changes of TU212 cells after treated with ferroptosis-induced agent erastin were detected by transmission electron microscope, cell counting kit-8(CCK-8), clone test, reactive oxygen species(ROS), malondialdehyde(MDA), glutathione(GSH), JC-1, RT-PCR and western blot. Exosomes were isolated from the supernatant of M0/M2 macrophages (M0-exos/M2-exos) and co-incubated with erastin-treated TU212 cells to detect the change of ferroptosis in cells of each group. The data were analyzed by SPSS software of version19.0. Results: GPX4 expression in LSCC tissues was significantly higher than that in adjacent noncancerous tissues (2.04±0.65 vs. 0.99±0.09, F=30.36, P<0.001), and was closely related to T stage and clinical stage (Ⅰ-Ⅱvs.Ⅲ-Ⅳ: 1.75±0.39 vs. 2.18±0.71, F=2.25, P<0.05; T1-2 vs. T3-4: 1.71±0.42 vs. 2.20±0.69, F=2.06, P<0.05). In TU212 cells treated with erastin, mitochondrial crest became smaller, membrane density increased, proliferation rate decreased, intracellular ROS level increased, mitochondrial membrane potential depolarized, GSH content decreased, intracellular MDA level increased and expressions of GPX4 mRNA and protein decreased. Change of M0 into M2 macrophages was induced by IL-4 stimulation. When erastin-treated TU212 cells were incubated with M2-exos, cell proliferation was partially restored and GPX4 expression was enhanced, and also with the recoveries of levels of ROS, MDA and GSH (all P<0.05). Conclusions: Ferroptosis is one of the cell death ways of LSCC. M2-exos may inhibit ferroptosis of LSCC cells.

Immunohistochemical detection of "ex novo" HLA-DR in tumor cells determines clinical outcome in laryngeal cancer patients.

There are controversial results about the role of "ex novo" HLA-DR expression by tumor cells and its correlation with the oncological outcomes. Unfortunately, little is known about HLA-DR expression in laryngeal cancer tumor cells. The main purpose of this retrospective study is to strengthen the usefulness of studying "ex novo" HLA-DR expression on tumor cells from primary laryngeal squamous cell carcinoma (LSCC) patients and investigate its correlation with clinical outcome. We analyzed HLA-DR expression by immunohistochemical analysis in 56 patients with LSCC. The "ex novo" HLA-DR expression on laryngeal cancer tumor cells, assessing non-neoplastic LSCC - adjacent tissue, and the association of HLA-DR expression (HLA-DR+) with clinical outcomes were investigated. HLA-DR+ tumor cells were detected in 18/56 LSCC patients (32.1%). All specimens of non-neoplastic laryngeal carcinoma-adjacent tissue resulted HLA-DR negative (HLA-DR-). A statistically significant association was observed between HLA-DR + and well differentiated tumors (G1) (p<0.001). The Kaplan-Meier method showed how HLA-DR+ is significantly associated with both a better disease specific survival (HLA-DR+=100% vs. HLA-DR-=77.4%; p=0.047) and a better relapse free survival (HLA-DR+=100% vs. HLA-DR-=72.3%; p=0.021). Cox regression univariate analysis for death of disease confirmed a higher HR for HLA-DR absence on the surface of epithelial tumor cell [HR:37.489; 95% CI:0.750-18730.776; p=0.253] and for high-grade (G3) tumors [HR:18.601; 95% CI:3.613-95.764; p<0.0001]. Our results confirm that MHC class II HLA-DR expression is activated in a sub-set of LSCC patients. Evaluation of HLA-DR expression in LSCC could be useful for prognosis and future approaches towards personalized therapy.

Loss of the MAF Transcription Factor in Laryngeal Squamous Cell Carcinoma.

MAF is a transcription factor that may act either as a tumor suppressor or as an oncogene, depending on cell type. We have shown previously that the overexpressed miR-1290 influences MAF protein levels in LSCC (laryngeal squamous cell carcinoma) cell lines. In this study, we shed further light on the interaction between miR-1290 and MAF, as well as on cellular MAF protein localization in LSCC. We confirmed the direct interaction between miR-1290 and MAF 3′UTR by a dual-luciferase reporter assay. In addition, we used immunohistochemistry staining to analyze MAF protein distribution and observed loss of MAF nuclear expression in 58% LSCC samples, of which 10% showed complete absence of MAF, compared to nuclear and cytoplasmatic expression in 100% normal mucosa. Using TCGA data, bisulfite pyrosequencing and CNV analysis, we excluded the possibility that loss-of-function mutations, promoter region DNA methylation or CNV are responsible for MAF loss in LSCC. Finally, we identified genes involved in the regulation of apoptosis harboring the MAF binding motif in their promoter region by applied FIMO and DAVID GO analysis. Our results highlight the role of miR-1290 in suppressing MAF expression in LSCC. Furthermore, MAF loss or mislocalization in FFPE LSCC tumor samples might suggest that MAF acts as a LSCC tumor suppressor by regulating apoptosis.

Sevoflurane promotes the apoptosis of laryngeal squamous cell carcinoma in-vitro and inhibits its malignant progression via miR-26a/FOXO1 axis

ABSTRACT Laryngeal squamous cell carcinoma (LSCC) is a laryngeal malignancy with a high mortality rates, and its treatment remains difficult. Sevoflurane is a surgical anesthesia which has anti-tumor effect. This investigation assessed the effects of LSCC cells treatment with Sevoflurane in vitro and in vivo. Hep-2 and Tu177 cells, human LSCC samples and BALB/C nude mice were used for result assessments. Cell viability, proliferation, migration and invasion were assessed via Cell Count Kit-8, wound healing assay and transwell invasion assay respectively. MiR-26a and FOXO1 expressions was examined by qRT-PCR. FOXO1, E-cadherin, N-cadherin and vimentin activities were examined by Western blotting. Moreover, animal experiments were performed to verify our findings in vitro. Lastly, miR-26a and FOXO1 expression levels in clinical samples were analyzed. According to the results, Sevoflurane decreased LSCC cells’ viability and even stimulated their apoptosis in vitro and in vivo. Moreover, it could reduce the migration, invasion and EMT. Mechanistically, sevoflurane could downregulate miR-26a expression and that miR-26a could negatively modulate FOXO1 activity. Thus, sevoflurane could increase FOXO1 activity. In the clinical samples, miR-26a expression was significantly upregulated, but FOXO1 was remarkably down-regulated and miR-26a expression in LSCC was linked with better prognosis. In conclusion, MiR-26a is increased and FOXO1 is reduced in human LSCC, Sevoflurane inhibits proliferation and mediates apoptosis of LSCC cells. Further, MiR-26a binds FOXO1 directly, and FOXO1 expression is down-regulated by Sevoflurane. Finally, Sevoflurane triggers LSCC cells apoptosis in vivo. Sevoflurane use to target miR-26a/FOXO1 may be a novel alternative for LSCC therapy.

OTX1 exerts an oncogenic role and is negatively regulated by miR129-5p in laryngeal squamous cell carcinoma

BackgroundOrthodenticle homeobox 1 (OTX1) is a transcription factor that plays an important role in various human cancers. However, the function of OTX1 in laryngeal squamous cell carcinoma (LSCC) is largely unknown. We aimed to explore the roles of OTX1 in LSCC and its possible molecular mechanism.MethodsThe expression levels of OTX1 were assessed in LSCC cell lines and tissue samples. We further examined the effect of OTX1 on LSCC progression. The upstream regulator of OTX1 was identified using a computer algorithm and confirmed experimentally.ResultsOTX1 was highly expressed in 70.7% (70/99) of LSCC tissue samples. The OTX1 expression in LSCC was significantly correlated with lymph node metastasis. High OTX1 expression in patients with LSCC was correlated with poor prognosis. Knockdown of OTX1 inhibited proliferation, colony formation, migration and invasion in LSCC cells. Knockdown of OTX1 inhibited tumor growth in a xenograft mouse model. Mechanistically, OTX1 might act as a direct target of miR-129-5p. OTX1 enhanced tumorigenicity and tumor growth both in vitro and in vivo.ConclusionsOur findings support that OTX1 is an oncogene in LSCC tumorigenesis and progression. Furthermore, OTX1 is a direct target of miR-129-5p in LSCC cells. Taken together, OTX1 is a promising diagnostic and therapeutic marker for LSCC.

Low expression of miR-20b-5p indicates favorable prognosis in laryngeal squamous cell carcinoma, especially in patients with non-infiltrated regional lymph nodes.

Tumor recurrence and distant metastasis are very common in laryngeal squamous cell carcinoma (LSCC). In this study, we examined the potential prognostic value of microRNA-20b-5p (miR-20b-5p), a component of the tumor-related miR-106a/363 cluster. Total RNA was purified from 105 tissue specimens resected from patients having undergone surgical treatment for primary LSCC. After in vitro polyadenylation and reverse transcription, a sensitive real-time quantitative polymerase chain reaction (qPCR) methodology was applied for the relative quantification of miR-20b-5p levels. Then, we proceeded with biostatistical analysis, seeking to assess the prognostic value of miR-20b-5p expression in LSCC. miR-20b-5p positivity constitutes a predictor of inferior DFS and OS in LSCC (P<0.001 and P=0.002, respectively). The significant prognostic value of miR-20b-5p expression status seems to be independent of tumor size, histological grade, and TNM stage, as revealed by the multivariate bootstrap Cox regression analysis. Kaplan-Meier survival analysis showed also that miR-20b-5p expression status can stratify LSCC patients with non-infiltrated regional lymph nodes (N0) into two subgroups with distinct prognosis (P=0.004 and P=0.004, respectively). The miR-20b-5p expression status is a promising molecular tissue biomarker in LSCC, with an independent prognostic value, and thus merits further validation in larger cohorts of patients.

Cyclin D1 Gene Numerical Imbalances in Laryngeal Squamous Cell Carcinoma: A Tissue Microarray Grid Based Analysis.

Background:Deregulation of critical proteins involved in cell cycle stability, such as cyclins, is a frequent genetic event in the development and progression of solid malignancies. Concerning laryngeal squamous cell carcinoma (LSCC), cyclin D1 oncogenic transformation leads to an aberrant protein expression and seems to affect the biological behaviour of the neoplasm. The aim of this study was to determine the correlation of cyclin D1 numerical imbalances with the corresponding protein expression levels in LSCCs. Material and Method:Using tissue microarray (TMA) technology, fifty (n=50) histologically confirmed primary LSSCs were cored at a diameter of 1.5 mm. Immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) analyses were applied. Concerning the screening process in CISH slides, a novel real-time reference and calibration grid platform was implemented. Results:Protein overexpression was observed in 22/50 (44%) cases; whereas, gene amplification was seen in 13/50 (26%) cases (p=0.02). Combined protein/ gene deregulation was associated with the stage of malignancy (p= 0.0014, p=0.001), whereas overall protein expression was strongly correlated with the grade of tumour (p= 0.001). Conclusion: Cyclin D1 gene amplification led to aberrant protein expression in LSCCs and it was also correlated with an aggressive biological behaviour. To best of our knowledge, this study was the first described grid based CISH analysis under conventional bright field microscopy for detecting gene numerical imbalances while providing a novel and accurate description for screening-mapping process in the entire slide area.

Comparison of TMA Technique and Routine Whole Slide Analysis in Evaluation of Proliferative Markers Expression in Laryngeal Squamous Cell Cancer.

Comparison of the expression of Ki-67, MCM3, 5, 7 and MTI/II proteins using immunohistochemistry (IHC) on whole section (WS) and tissue microarray (TMA) of laryngeal squamous cell carcinoma (LSCC) samples. A total of 51 archival paraffin blocks of LSCC were used. TMAs were prepared from 1.5 mm core punches. IHC reactions were performed using antibodies against Ki-67, minichromosome maintenance proteins (MCM3, 5, 7) and metallothionein (MTI/II). Spearman rank correlation test revealed moderate positive correlation in the case of Ki-67: WS vs. TMA (r=0.38, p=0.07) and strong positive correlation in regard to the rest of tested markers: MCM3, WS vs. TMA (r=0.49, p=0.0004); MCM5, WS vs. TMA (r=0.61, p<0.0001); MCM7, WS vs. TMA (r=0.59, p<0.0001); MTI/II, WS vs. TMA (r=0.66, p<0.0001). Mann Whitney U-test showed no significant differences in the case of Ki-67 and MCM5. Moreover, Bland-Altman test showed a low level of bias in regard to Ki-67, WS vs. TMA and MCM5, WS vs. TMA. TMA may be an effective and reliable method of assessment of Ki-67 and MCM5 expression in LSCC.

Correlation Between the NLRP3 Inflammasome and the Prognosis of Patients With LSCC.

Background: NLRP3 inflammasome is an inflammatory mediator. The expression of NLRP3 inflammasome is associated with the development of various tumors and is closely related to the prognosis of tumors. However, the role of NLRP3 inflammasome in laryngeal squamous cell carcinoma (LSCC) remains unclear. This study aim to investigate the influence of NLPR3 inflammasome expression in LSCC, and especially the NLRP3 inflammasome expression level and the prognosis of LSCC after surgery in a Chinese population.Methods: We used quantitative real-time PCR and immunohistochemical (IHC) staining to calculate the mRNA (20 patients, fresh tissue) and protein expression (104 patients, paraffin tissue microarray) levels of the NLRP3 inflammasome (NLRP3/IL-18/IL-1β/ASC/caspase-1), respectively. We also analyzed the relationship between NLRP3 inflammasome expression levels and LSCC cancer tissues compared with adjacent normal tissues and the clinical features of LSCC. Kaplan–Meier survival curves of overall survival (OS) and disease-free survival (DFS) in LSCC patients were compared and analyzed under different expression levels of the NLRP3 inflammasome.Results: Our results indicated that the mRNA expression of the NLRP3 inflammasome was higher in LSCC cancer tissues compared with adjacent normal tissues (p < 0.001). The IHC staining score also demonstrated that the expression of the NLRP3 inflammasome was higher than in the adjacent normal tissues (p < 0.001). The NLRP3 inflammasome expression also exhibited a close relationship with the clinicopathological characteristics (especially the stage of LSCC) of LSCC. Univariate Cox regression analysis and multivariate Cox regression analysis revealed that both NLRP3 and IL-1β had an increased risk of LSCC progression (p < 0.05). The Kaplan–Meier log rank test (OS and DFS) demonstrated that high expression of NLRP3/IL-18/IL-1β/ASC was statistically different than the low expression group (p < 0.05) of LSCC patients after surgery.Conclusion: The high expression group of the NLRP3 inflammasome (NLRP3/IL-18/IL-1β/ASC) had a poorer prognosis (OS and DFS) than the low expression group of LSCC patients 5 years after surgery. The NLRP3 inflammasome (NLRP3/IL-18/IL-1β/ASC) may be used as an auxiliary indicator to predict LSCC patient prognosis after surgery.

Study on the mechanism of miR-497-induced laryngeal squamous cell carcinoma growth inhibition by targeting CDK6

Objective: To study the effects of miR-497 and CDK6 on the growth of laryngeal squamous cell carcinoma (LSCC). Methods: The expressions of CDK6 mRNA in fresh LSCC specimens, the adjacent normal mucosa of LSCC, and cell lines of LSCC were detected with quantitative real time polymerase chain reaction, pcDNA3.1(+) CDK6 plasmids were respectively transfected into the LSCC cells, and MTT assay and clone formation assay were performed to evaluate the growth of LSCC cells. Flow cytometry was employed for cell cycle analysis. SPSS17.0 software was used to analyze the data. Results: CDK6 was highly expressed in LSCC(t=14.01, P=0.009) and the overall survival rate of the patients with high CDK6 expression was less than that with low CDK6 expression, with a significant difference (HR=3.236, P<0.001). Double luciferase reporter gene analysis showed that fluorescence activity in wild type CDK6 group was significantly different from that in control group (P<0.01), while there was no significant difference in the fluorescence activity between mutant CDK6 group and control group (P>0.05). A(490) values were respectively 0.42±0.14 (Mean±SD) in siRNA Hep-2 group, 0.51±0.13 in siRNA TU-212 group; 0.98±0.16 in control Hep-2 group and 1.17±0.20 in control TU-212 group. Colonies were 55±4 in siRNA Hep-2 group, 51±3 in siRNA TU-212 group, 108±6 in control Hep-2 group and 105±7 in control TU-212 group, namely, cell growth and clone formation ability in CDK6 siRNA group were significantly lower than those in the control group. Cells cycle was blocked in G0/G1 phase (G0/G1: 65.20%±10.12% in siRNA Hep-2 group; 63.42%±8.97% in siRNA TU-212 group; 45.31%±7.55% in control Hep-2 group; and 42.37%±7.28% in control TU-212 group), and cells decreased obviously in S phase (S: 25.39%±5.51% in siRNA Hep-2 group; 27.21%±5.43% in siRNA TU-212 group; 42.87%±6.85% in control Hep-2 group; and 44.76%±7.02% in control TU-212 group). Compared with miR-497 group, cell growth and clone formation ability in miR-497/CDK6 group were partly restored (all P<0.05). Conclusions: CDK6 expression in LSCC is upregulated, functioning as an oncogene. High expression of CDK6 is a predictor for poor prognosis. miR-497, functioning as a tumor suppressor gene, inhibits the growth of LSCC by targeting CDK6.

Nuclear expression of onco-suppressors nm23-H1 and maspin are associated with lower recurrence rate in laryngeal carcinoma.

The main aim of the study was to preliminarily investigate the possibly related role of nuclear onco-suppressors maspin and nm23-H1, a metastasis suppressor, in laryngeal squamous cell carcinoma (LSCC). Maspin expression pattern and nuclear nm23-H1 expression were ascertained in 62 consecutive LSCCs. Recurrence rate was significantly lower in patients with a nuclear maspin pattern of expression; nuclear nm23-H1 expression was significantly lower in patients who experienced disease recurrence. Disease free survival (DFS) was significantly longer in patients with maspin nuclear pattern or with nuclear nm23-H1 expression ≥10%. A significant association was found between nuclear nm23-H1 expression and maspin pattern of expression in LSCC. KNN discriminant analysis considered N status, maspin sub-cellular localization and nuclear nm23-H1 expression. The selected variables' accuracy in terms of relapse was 82%. Positive predictive accuracy was 100%, and negative predictive accuracy 79%. Nuclear nm23-H1 expression and maspin pattern, also in association, show promise as recurrence indicators in LSCC. Further studies are needed to shed more light on the nm23-H1 mechanism of action in LSCC and thus find ways to restore nm23-H1 loss. These preliminary findings suggest that re-activating maspin functions might represent an important goal in the treatment of advanced LSCC.

CDR1as is overexpressed in laryngeal squamous cell carcinoma to promote the tumour's progression via miR-7 signals.

To investigate the roles played by the circular RNA (circRNA) molecule ciRS-7 (CDR1as) and tumour suppressor miRNA-7 (miR-7) in laryngeal squamous cell carcinoma (LSCC). Specimens of LSCC tissue (n=30) and corresponding relative normal tissue (n=30) were collected to determine their levels and clinical significance of CDR1as/mir-7 expression. The CDR1as and miR-7 were overexpressed in LSCC cells to investigate its function and mechanism in vitro and in vivo. Patients with high TNM stages, poorly differentiated tumours, lymph node metastases and poor prognosis had high CDR1as levels but low miR-7 levels. CDR1 expression was negatively associated with miR-7 expression in LSCC. Overexpression of CDR1as in vitro enhanced cell vitality, and promoted the proliferation, migration, and invasion of two LSCC cell lines (Hep2 and AMC-HN-8.) However, these effects could be abrogated by knockdown of CDR1as or the forced expression of miR-7. Mechanistically, overexpressed CDR1 molecules functioned as miR-7 sponges and upregulated the key targets of miR-7, CCNE1, and PIK3CD in Hep2 and AMC-HN-8 cells. In vivo studies demonstrated the tumourigenic role of CDR1as. Overexpression of CDR1as alone promoted tumour growth and increased expression of the proliferation indices ki-67, CCNE1, and PIK3CD. Although the tumour suppressor miR-7 effectively inhibited the tumour growth, this effect could be counteracted by co-treatment with CDR1as in vivo. CDR1as is an oncogene that promotes LSCC progression by regulating miR-7 signals.

The effect of miR-497 on laryngeal squamous cell carcinoma invasion through modulating PlexinA4

Objective: To study the roles of miR-497 and PlexinA4 in the progression of laryngeal squamous cell carcinoma. Methods: The expressions of miR-497 and PlexinA4 in fresh tumor specimens and adjacent normal mucosa tissues as well as in cell lines of laryngeal squamous cell carcinoma (LSCC) were detected with qRT-PCR and immunohistochemistry. The association of miR-497 and PlexinA4 expressions with clinicopathologic factors and their prognostic values in LSCC were evaluated PlexinA4 siRNA and pcDNA3.1 (+ )/PlexinA4 plasmid were transfected into the LSCC and measured by Transwell to evaluate their effect on the invasion of LSCC. Results: miR-497 was low expression in LSCC, which related to pathological differentiation, while PlexinA4 mRNA was high expression in LSCC. Kaplan-Meier method showed that the prognosis of patients with high miR-497 expression was better than that of patients with low miR-497 expression (χ(2)=10.342, P=0.001); . Cox regression analysis showed that miR-497 was an independent prognostic factor for LSCC. The double luciferase reporter gene showed that the variation of the fluorescence activity of wild type PlexinA4 was significantly different from that of the control group (P<0.01). In Hep-2 and TU212 cell line, the number of cells with PlexinA4 siRNA passing through the compartments was 70.00±10.85 and 85.00±6.45, significantly higher than control (F values were 30.251 and 23.936, both P<0.05), the number of cells with pcDNA3.1 (+ ) /PlexinA4 was 170.56±11.95 and 142.00±10.43, also significantly less than control (F values were 35.104 and 29.643, both P<0.05). Conclusion: The expression of miR-497 in LSCC is decreased, indicating poor prognosis, which is as an independent risk factor for prognosis of LSCC. miR-497 may modulate LSCC invasion through PlexinA4.