Novel defined N7-methylguanosine modification-related lncRNAs for predicting the prognosis of laryngeal squamous cell carcinoma

Objective:Through integrated bioinformatics analysis,the goal of this work was to find new,characterised N7-methylguanosine modification-related long non-coding RNAs(m7G-lncRNAs)that might be used to predict the prognosis of laryngeal squamous cell carcinoma(LSCC).Methods:The clinical data and LSCC gene expression data for the current investigation were initially retrieved from the TCGA database&sanitised.Then,using co-expression analysis of m7G-associated mRNAs&lncRNAs&differential expression analysis(DEA)among LSCC&normal sample categories,we discovered lncRNAs that were connected to m7G.The prognosis prediction model was built for the training category using univariate&multivariate COX regression&LASSO regression analyses,&the model’s efficacy was checked against the test category data.In addition,we conducted DEA of prognostic m7G-lncRNAs among LSCC&normal sample categories&compiled a list of co-expression networks&the structure of prognosis m7G-lncRNAs.To compare the prognoses for individuals with LSCC in the high-&low-risk categories in the prognosis prediction model,survival and risk assessments were also carried out.Finally,we created a nomogram to accurately forecast the outcomes of LSCC patients&created receiver operating characteristic(ROC)curves to assess the prognosis prediction model’s predictive capability.Results:Using co-expression network analysis&differential expression analysis,we discovered 774 m7G-lncRNAs and 551 DEm7G-lncRNAs,respectively.We then constructed a prognosis prediction model for six m7G-lncRNAs(FLG−AS1,RHOA−IT1,AC020913.3,AC027307.2,AC010973.2 and AC010789.1),identified 32 DEPm7G-lncRNAs,analyzed the correlation between 32 DEPm7G-lncRNAs and 13 DEPm7G-mRNAs,and performed survival analyses and risk analyses of the prognosis prediction model to assess the prognostic performance of LSCC patients.By displaying ROC curves and a nomogram,we finally checked the prognosis prediction model's accuracy.Conclusion:By creating novel predictive lncRNA signatures for clinical diagnosis&therapy,our findings will contribute to understanding the pathogenetic process of LSCC.

Expression of Hypoxia Inducible Factor-1α and Its Relationship to Apoptosis and Proliferation in Human Laryngeal Squamous Cell Carcinoma

Summary: To investigate the expression of hypoxia inducible factor-1 alpha (HIF-1α) and its relationship to apoptosis and proliferation in laryngeal squamous cell carcinoma (LSCC), immunohistochemical method was used to detect the expression of HIF-1α and PCNA. Tunnel technique was used to detect in situ cell apoptosis in LSCC. Our results showed that the expression of HIF-1α was related to the clinical stages of cancer and lymph node metastasis (P<0.05). The relationship between HIF-1α and PCNA was statistically significant (P<0.05) and no relationship was found between HIF-1α and apoptosis (P>0.05) It is concluded that HIF-1α plays a role in the carcinogenesis of laryngeal carcinoma and is correlated with proliferation, but bears no relationship with the apoptosis of tumor cells in LSCC.

Expression of Vascular Endothelial Growth Factor and Cyclooxygenase-2 in Laryngeal Squamous Cell Carcinoma and Its significance

n order to study the expressions of vascular endothelial growth factor （VEGF） and cyclooxygenase-2 （COX-2） in human laryngeal squamous cell carcinoma （LSCC） and its significance, the expression of VEGF mRNA and COX-2 mRNA in 62 cases of LSCC and 54 adjacent noncancerous laryngeal tissues and 9 normal human laryngeal mucous tissues was detected by using techniques of semi-quantitative RT-PCR. It was found that the expression level of VEGF and COX-2 mRNA was significantly increased in LSCC as compared with that in the normal human laryngeal mucous tissues （both P〈0. 01）, and the expression level of VEGF and COX-2 mRNA were significantly increased in stage Ⅲ＋ Ⅳtissues of LSCC as compared with the stage Ⅰ ＋ Ⅱ tissues of LSCC （P 〈0.01）. There was a high positive correlation between VEGF and COX-2 expression in LSCC （r= 0. 756,P〈0.01）. These data raise the possibility that VEGF and COX-2 may play key roles in the growth, invasion and metastasis of LSCC.

Detection of Human Papilloma Virus Type 16 E6 mRNA in Laryngeal Squamous Cell Carcinoma by In Situ Hybridization

Objective：Laryngeal squamous cell carcinoma（LSCC） is a common malignant tumor in Northeast China and is frequently associated with well-established risk factors like smoking and alcohol abuse.Human papilloma virus（HPV） is an epitheliotropic oncogenic virus that has been detected in a variety of head and neck tumors including LSCC.This retrospective study was to investigate the prevalence of HPV infection in patients with LSCC.Methods：In situ hybridization was performed in 99 patients with LSCC to detect the expression of HPV-16 E6 mRNA.Results：The positive rate of HPV16 E6 mRNA was 36.36%（36/99） in laryngeal squamous cell carcinoma（LSCC）,whereas only 3 of 50（6%） specimens of the normal laryngeal mucosa as a control group showed positive results（P0.05）.Additionally,there was no corelation between HPV16 and age,gender,clinical stage,nodal status and tumor site（P0.05）.Conclusion：The results suggest that the increased prevalence of HPV infection compared to normal laryngeal mucosa and the fact that high-risk HPV types（especially type 16） were the most frequently identified do not allow the exclusion of HPV as a risk factor in laryngeal squamous cell carcinoma.However,their clinical value remains to be further investigated.

MiR-194 functions as a tumor suppressor in laryngeal squamous cell carcinoma by targeting Wee1

OBJECTIVE To investigate the clinical and functional association of mi R-194 in human laryngeal squamous cell carcinoma(LSCC).METHODS Cell growth was measured by MTT assay.Cel cycle distribution was detected using PI staining by flow cytometric analysis.Cell migration and invasion were examined by wound healing assay and transwell assay.The 3′-UTR activity was detected by luciferase assay.The expression level of proteins and mR NA were analysed by Western blotting,immunohistochemistry and q RT-PCR.Mouse xenograft model was established to observe the tumor growth in vivo.RESULTS The expression level of miR-194 is significantly lower in clinical LSCC tissues compared with normal tissues,and is correlated with lymph node metastasis and TNM stage.Kaplan-Meier analysis shows that high miR-194 expression predicts a favorable outcome for LSCC patients.Functional assays show that enforced expression of mi R-194 inhibits the growth,migration,invasion and drug-resistance of LSCC cells.Moreover,Wee1 is identified as a novel functional target of mi R-194.Exogenous expression of Wee1 protein in mi R-194-over expressing cells partially reverses the suppressive effects of mi R-194 on LSCC cells.In addition,Wee1 was abnormally overexpressed in clinical LSCC tissues,and its protein levels were inversely correlated with miR-194 expression.High Wee1 protein level was also associated with TNM stage,lymph node metastasis and poor prognosis.CONCLUSION Our study provides new sights into the role of miR-194/Wee1 axis in LSCC,and suggests a novel miR-194/Wee1-based clinical intervention target for LSCC patients.

A CORRELATIVE STUDY OF Ki67 AND VASCULAR ENDOTHELIAL GROWTH FACTOR AND THEIR VALUE IN LARYNGEAL SQUAMOUS CELL CARCINOMA

Objective： To study the correlation between Ki67 and vascular endothelial growth factor（VEGF） and the significance of their expression in laryngeal squamous cell carcinoma（LSCC）. Methods： The expressions of Ki67 and VEGF in 40 cases of LSCC and 5 cases of normal laryngeal mucosa were examined by immunohistochemical staining. Results： The expression levels of Ki67 and VEGF in LSCC tissue were higher than in normal laryngeal mucosa （Ki67： P〈0.001, VEGF： P〈0.001）. The two indexes＇ levels in patients of different age or different sex had no significant difference （P〉0.05）. They were higher in LSCC with metastasis of lymph nodes than in patients without metastasis （Ki67： P=0.034, VEGF： P=0.006）. The expressions of the two genes elevated correspondingly along with the development of LSCC T stage （P〈0.05）. In addition, correlation analysis indicated that the expression of Ki67 had a positive correlation with VEGF in LSCC（r=0.823, P〈0.01）. Conclusion： Ki67 and VEGF are objective indexes for the biological behavior of LSCC, and they might be helpful to the diagnosis, treatment and prognosis of laryngocarcinoma.

Overexpression of interleukin-17 in tumor-associated macrophages is correlated with the differentiation and angiogenesis of laryngeal squamous cell carcinoma

Background Interleukin-l7 （IL-17）, which exerts strong pro-inflammatory effects, has emerged as an important mediator in inflammation-associated cancer. The aim of this study was to clarify the relationship between IL-17 and tumor associated macrophages （TAMs）, and the correlation of the microvessel density in the development of laryngeal squamous cell carcinoma （LSCC）.Methods Histopathological observations and immunohistochemistry staining for IL-17, CD68, and CD34 were performed on 72 specimens （32 cases of LSCC, 20 cases of adjacent tissues of carcinoma as controls, and 20 cases of chronic hypertrophic laryngitis）. Double immunohistochemical staining was done to determine which cells expressed IL-17. Real-time quantitative PCR determined the mRNA expression of IL-17. ELISA was used to detect the expression of the serum level of IL-17 in the three groups.Results The inflammation response had increased in LSCC. Overexpression of IL-17 and CD68 protein were seen in LSCC （P 〈0.01）. The expression of IL-17 was different between well and poorly differentiated LSCC （P 〈0.01）. The IL-17 expressing cells were mainly located in macrophages （CD68＋/IL17＋） as demonstrated by double immunohistochemical staining. IL-17 expression significantly correlated with high microvessel density （CD34＋） in LSCC （P 〈0.05）. Relatively higher mRNA expression levels of IL-17 were seen in LSCC compared to the controls （P 〈0.05）. The serum expression of IL-17 was similar among the three groups （P 〉0.05）.Conclusion IL-17 was expressed by TAMs, and IL-17 may significantly correlate to the differentiation and angiogenesis in the development of LSCC.

Vasculogenic mimicry is a key prognostic factor for laryngeal squamous cell carcinoma： a new pattern of blood supply

Background Recurrence and local lymph node metastasis affected the prognosis of patients with laryngeal cancer. The aim of this study was to analyze clinical and pathological significance of vasculogenic mimicry （VM） in laryngeal squamous cell carcinoma （LSCC） and evaluate its contribution to prognosis. Methods Data of 168 cases of LSCC were reviewed retrospectively to reveal clinical pathology and prognostic significance of VM. CD31 and periodic acid-Schiff double staining was used to identify VM. Results VM in LSCC contributed to lymph node metastasis （P=0.003） and clinical progression. VM correlated to histopathology grade （P=0.001） of LSCC. VM was an adverse prognostic factor for both disease-specific survival （P=0.039） and metastasis-free survival （P=0.042） by univariate survival analyses. And it was an independent prognostic factor for only disease-specific survival （P=0.003） by multivariate survival analyses. Conclusions VM existed in LSCC. LSCC with VM has more Dotential to invasion and metastasis.

Prognostic significance of y-H2AX in laryngeal squamous cell carcinoma after surgery

Background The clinical significance of Y-H2AX in laryngeal squamous cell carcinoma （LSCC） has not yet been established. This study was performed to assess the expression of nuclear y-H2AX in benign and malignant laryngeal lesions and to assess its clinicopathological significance. Methods A total of 70 LSCC tumor-normal tissue paired samples were evaluated for y-H2AX expression using immunohistochemical staining. Their expression was correlated with different clinicopathological parameters. Results Nuclear Y-H2AX expression was frequently detected in LSCC tissues （P 〈0.001）. High nuclear Y-H2AX levels were not associated with any clinicopathological characteristics of LSCC （P 〉0.05）. Univariate Kaplan-Meier analysis showed that positive nuclear Y-H2AX expression was associated with a decreased overall survival （P=0.017）. Multivariate Cox regression analysis showed that nuclear Y-H2AX expression was an independent risk factor for overall survival. Conclusion The expression of nuclear Y-H2AX might be closely related to the prognosis of LSCC. Chin Med J 2014;127 （14）： 2664-2667

Detailed deletion mapping of loss of heterozygosity on 9p13-23 in laryngeal squamous cell carcinoma by microsatellite analysis

Background This study was designed to investigate the hot spots of microsatellite loss of heterozygosity (LOH) on 9p13-23 in laryngeal squamous cell carcinoma and to find out the correlation between the incidence of microsatellite LOH and the clinicopathological parameters Methods Tumor tissues were obtained from paraffin embedded sections with microdissection Genomic DNA was extracted from tumor tissues and peripheral blood lymphocytes with the phenol-chloroform Polymerase chain reaction (PCR) amplification and denaturing gel electrophoresis were carried out in a set of 42 squamous cell carcinoma (SCC) of larynx and corresponding peripheral blood lymphocytes using 13 highly polymorphic microsatellite markers on 9p13-23 The correlation was analyzed between microsatellite LOH at the high frequency on 9p13-23 and clinicopathological parameters in the patients with squamous cell carcinoma of larynx KH*2/5DResults Of the 42 laryngeal cancers, 41 (97 6%) showed LOH in at least one of the microsatellite markers tested on 9p13-23 The most frequently deleted marker was D9S162 in 17 of the 19 (89 5%) informative samples The marker D9S171, which is located on 9p21, had LOH detected in 12 of the 15 informative cases (80 0%) LOH at the D9S1748 marker (closest to the p16 gene locus) was detected in 18 of the 36 informative cases (50 0%) Allelic deletion mapping revealed two minimal regions of LOH encompassing markers D9S161-D9S171 on 9p21 and IFNA-D9S162 on 9p22-23 Multiple LOH (≥4) on 9p21-23 was found more frequently in the patients under 60 years, with supraglottic SCC or cervical lymph node metastasis than those over 60 years, with glottic SCC or without cervical lymph node metastasis ( P <0 01 or 0 01, 0 05, respectively) On the contrary, there was no correlation between T stages or pathologic classification and the frequency of LOH on 9p21-23 in 42 SCC of Larynx Conclusions These findings imply the presence of at least two putative tumor suppressor genes on 9p13-23 in laryngeal SCC Multiple genetic alterations are probably implicated in supraglottic SCC with cervical lymph node metastasis in younger patients

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 upreg-ulated,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 ran-domly 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 malig-nant phenotypes through regulation of LSM6.We anticipate that HMGB1-LSM6 could be a putative therapeutic target for LSCC.

Tumor necrosis factor a accelerates Hep-2 cells proliferation by suppressing TRPP2 expression

TRPP2, a Ca^(2+)-permeable non-selective cation channel, has been shown to negatively regulate cell cycle, but the mechanism underlying this regulation is unknown. Tumor necrosis factor a(TNF-α) is a proinflammatory cytokine extensively involved in immune system regulation, cell proliferation and cell survival. However, the effects and mechanisms for the role of TNF-αin laryngeal cancer remain unclear. Here, we demonstrated using western blot analyses and intracellular Ca^(2+) concentration measurements that TNF-α treatment suppressed both TRPP2 expression and ATP-induced Ca^(2+) release in a laryngeal cancer cell line(Hep-2). Knockdown of TRPP2 by a specific siRNA significantly decreased ATP-induced Ca^(2+) release and abolished the effect of TNF-α on the ATP-induced Ca^(2+) release. TNF-α treatment also enhanced Hep-2 cell proliferation and growth, as determined using cell counting and flow cytometry cell cycle assays. Moreover, TNF-α treatment down-regulated phosphorylated protein kinase R-like endoplasmic reticulum kinase(p-PERK) and phosphorylated eukaryotic translation initiation factor(p-eIF2α)expression levels, without affecting PERK and eIF2 a expression levels in Hep-2 cells. We concluded that suppressing TRPP2 expression and TRPP2-mediated Ca^(2+) signaling may be one mechanism underlying TNF-α-enhanced Hep-2 cell proliferation.These results offer new insights into the mechanisms of TNF-α-mediated laryngeal cancer cell proliferation, and provide evidences showing a potential role of TNF-α in the development of laryngeal cancer.

Diagnostic accuracy of MRI and PET/CT for neck staging prior to salvage total laryngectomy

Aim:Lymph node(LN)metastases are associated with poor outcomes in patients with recurrent larynx squamous cell carcinoma(LSCC).Neck dissection(ND)is therefore commonly performed along with salvage total laryngectomy(STL).Here,we assess the rate of occult LN metastases and the diagnostic value of MRI and PET/CT for detecting them in recurrent LSCC.Methods:This retrospective study included patients with recurrent LSCC after primary(chemo)radiotherapy[(C)RT]who were re-staged by MRI and/or PET/CT and treated with STL and ND between 2004 and 2019.The histopathology of ND samples was used as the reference standard.Results:Forty-one patients were included.The prevalence of occult metastases in MRI-negative and PET/CT-negative neck nodes was between 3.2%and 6.1%.Negative predictive values of neck node re-staging were 93.9%for MRI,96.8%for PET/CT,and 96.2%for MRI and PET/CT combined.Conclusion:Both MRI and PET/CT afforded good negative predictive values for nodal staging in patients with recurrent LSCC after(C)RT prior to STL.In selected patients,these radiological modalities,particularly PET/CT,could help to avoid unnecessary surgery to the neck and its associated morbidity.