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.

Relation between the Expression of K-ras in Hep-2 Cells and Development of Laryngeal Carcinoma~*

Objective： To investigate the expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and its significance for establishing a solid foundation for further study of the relationship between human laryngeal squamous cell carcinoma and K-ras gene point mutations. Methods： The expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and human pancreatic carcinoma cell lines （MIAPaCa-2） was detected by using RT-PCR. Results： The expression of K-ras mRNA in Hep-2 and MIAPaCa-2 was strong and positive. Conclusion： The expression of K-ras mRNA in human laryngeal squamous cell carcinoma cell lines （Hep-2） is positive. Development of laryngeal carcinoma might be related to the activation of K-ras gene point mutation.

The Prognostic Value of Pathological and Molecular Margins Marked by p53 and eIF4E in Laryngeal Carcinoma

Objective: To study the prognostic value of the pathological margin and molecular margin marked by eIF4E and P53 protein in laryngeal carcinoma. Methods: The prognostic value of pathological and molecular margin was studied in 253 cases and 67 cases respectively, the latter were pathological negative margin chosen from the former. Immunohistochemisty was used to detect the expression of eIF4E and p53 proteins. Results: The rate of pathological, p53 and eIF4E positive margins was 20.2%, 19.4% and 32.8% respectively. The recurrent rate of those with positive margins was higher than that of negative margins, which including pathological margin (70.6% vs 35.1%, P =0.0000), p53 margin (69.2% vs 33.3%, P =0.018) and eIF4E margin (63.6% vs 28.9%, P =0.018); The survival rate of those with negative margins was higher than those with positive margins, including pathological margin (the 5-year cumulative survival rate was 37.52% and 64.37% respectively, P =0.0023), p53 margin (the 5-year cumulative survival rate was 24.62% and 75.69% respectively, P =0.0012) and eIF4E margin (the 5-year cumulative survival rate was 43.31% and 77.52% respectively, P =0.0006). Conclusion: The prognosis of those with both pathological and molecular positive margins was worse than that of the negative margins; Both the eIF4E and p53 were useful markers to pick out the poor prognostic patients from those with pathological negative margin, and the former seemed to be more potential.

Regulation of the cell cycle gene, BTG2, by miR-21 in human laryngeal carcinoma

MicroRNAs are short regulatory RNAs that negatively modulate gene expression at the post-transcriptional level, and are deeply involved in the pathogenesis of several types of cancers. To investigate whether specific miRNAs and their target genes participate in the molecular pathogenesis of laryngeal carcinoma, oligonucleotide microarrays were used to assess the differential expression profiles of microRNAs and mRNAs in laryngeal carcinoma tissues compared with normal tissues. The oncogeuic miRNA, microRNA-21 （miR-21）, was found to he npregulated in laryngeal carcinoma tissues. Knockdown of miR-21 by specific antisense oligonucleotides inhibited the proliferation potential of HEp-2 cells, whereas overexpression of miR-21 elevated growth activity of the cells, as detected by the colony formation assay. The cell number reduction caused by miR-21 inhibition was due to the loss of control of the G1-S phase transition, instead of a noticeable increase in apoptosis. Subsequently, a new target gene of miR- 21, BTG2, was found to be downregulated in laryngeal carcinoma tissues. BTG2 is known to act as a pan-cell cycle regulator and tumor suppressor. These findings indicate that aberrant expression of miR-21 may contribute to the malignant phenotype of laryngeal carcinoma by maintaining a low level of BTG2. The identification of the oneogenic miR-21 and its target gene, BTG2, in laryngeal carcinoma is potentially valuable for cancer diagnosis and therapy.

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 PAPILLOMAVIRUS L1 -16 AND -18 DNA AND EPSTEIN-BARR VIRUS DNA IN LARYNGEAL CARCINOMA

Objective: To look for the further evidence for HPV L1 HPV16 E6, HPV 18 E6 and EBV as carcinogenic factors in laryngeal carcinoma. Method: we examined representative numbers of specimens from laryngeal cancer with highly sensitive PCR technique for the presence of HPV L1 and high-risk types HPV16 E6, HPV18 E6 and EBV LMP1. Results: Using PCR detection, 7.3% samples were HPV L1 positive, 52.03% were HPV16 E6 positive, 30.89% were HPV18 E6 positive and 9.13% were EBV LMP1 positive. The low incidence of HPV L1 and high incidence of HPV-16 E6 and HPV18 E6 genes suggest that HPV might be integrated into tumor cells. Our results support a role of HPV-16 and HPV-18 infection in the pathogenesis of laryngeal carcinoma in China. Conclusion: Integration of E6 into host genome and stable expression of these genes may be associated with the carcinogenesis of laryngeal carcinoma. HPV-16 and HPV-18 may synergistically function on the pathogenesis of laryngeal carcinoma. Our results suggest an association of laryngeal carcinogenesis and infection with the high-risk HPV types 16, HPV 18 and EBV.

CORRELATION ANALYSIS BETWEEN STK15 GENE AND LARYNGEAL CARCINOMA

To explore the relationship between STK15 gene abnormal expression and laryngeal carcinoma. Methods: Tumor tissues and matched normal tissues were taken from 55 LSCC patients. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect STK15 expression in 110 specimens. Results: In 38 of the 55 cases (69.1%), the STK15 expression at the mRNA levels was higher than that of the paired normal tissue. The ratio of ADV (average density value) of STK15 gene to ADV of b-actin gene was 1.220.49 in the cancer tissue, and 0.990.54 in the paired normal tissue with a significant difference (t=4.539, P<0.01). Conclusion: There was obvious association between the STK15 overexpression and laryngeal carcinoma. It may serve as an alternative mechanism of activating the pathogenesis of human laryngeal squamous cell carcinoma.

Promoter Hypermethylation of DNA Repair Gene MGMT in Laryngeal Squamous Cell Carcinoma

The relationship between hypermethylation of CpG islands in the promoter regions of O^6- methylguanine DNA methyhransferase （MGMT） genes and laryngeal squamous cell carcinoma was explored. Methylation-specific PCR and semi-quantitative RT-PCR were used to study the promoter methylation and mRNA expression of the MGMT gene in laryngeal carcinoma tissues, tissues adjacent to the tumor and normal laryngeal tissues. Hypermethylation of MGMT gene was detected in 16 samples of 46 （34.8 %） laryngeal squamous cell carcinoma samples. However, the MGMT hypermethylation was not detected in all tissues adjacent to the tumors and normal tissues. No significant difference in MGMT gene hypermethylation was found in samples with different histological grades （χ^2= 3. 130, P=0. 077） or in samples from patients with different TNM status （χ^2= 3. 957, P=0. 138）. No expression of MGMT mRNA was detected in all hypermethylated laryngeal carcinoma tissues. The expression of MGMT mRNA was detected in all unmethylated laryngeal carcinoma tissues, tissues adjacent to the tumors and normal tissues. It suggests that MGMT gene promoter hypermethylation is associated with MGMT gene transcription loss in laryngeal carcinoma tissues and possibly plays an important role in carcinogenesis of laryngeal tissues.

Correlation between Survivin Expression and Laryngeal Carcinoma:A Meta-analysis

In order to provide evidence for evidence-based medicine in the treatment and prognosis of laryngeal cancer in China, the meta-analysis electronically retrieved the case-control studies published in China about the Survivin expression and its association with clinical pathological features in the tissues of laryngeal carcinoma. The results showed that a total of 25 case-control studies were finally included with 1333 cases of laryngeal cancer and 528 cases of controls. The difference in the expression of Survivin between the two groups was statistically significant [OR=18.34, 95% CI（11.82, 28.47）, P〈0.00001]. The difference in the expression of Survivin between laryngeal carcinoma patients with lymph node metastasis or not was statistically significant [OR=0.25, 95% CI（0.17, 0.37）, P〈0.00001]. The expression of Survivin in clinical Ⅰ–Ⅱ stage group was significantly lower than in the clinical stage Ⅲ–Ⅳ group [OR=0.24, 95% CI（0.18, 0.32）, P〈0.00001]. The expression of Survivin in patients with low/medium differentiation was significantly lower than that in those with high differentiation [OR=0.33, 95% CI（0.26, 0.43）, P〈0.00001]. The difference in the expression of Survivin among different T stages of laryngeal carcinoma was statistically significant [OR=0.35, 95% CI（0.21, 0.58）, P〈0.00001]. In conclusion, Survivin may play an important role in the occurrence and development of laryngeal carcinoma, and its high expression is related to the poor prognosis of patients with laryngeal cancer.

Neoadjuvant chemotherapy with pingyangmycin can inhibit the amplification and metastasis of laryngeal squamous cell carcinoma

Objective:To evaluate the short-term effects of neoadjuvant chemotherapy with pingyangmycin(PYM)in the treat-ment of laryngeal squamous cell carcinoma.Methods:24 cases of laryngeal squamous cell carcinoma were treated with PYM before the operation,and the surgeries were undergone within one week after neoadjuvant chemotherapy.PCNA,p53,Bcl-2 and CD44v6 were detected in the specimens of tumor,retreated tumor and normal tissue using immunohistochemical methods.Results:Apoptosis could be detected more often in specimens with tumor and retreated tumor after chemotherapy than that before.The expression of PCNA,p53,Bcl-2 and CD44v6 in tumor tissue after neoadjuvant chemotherapy with PYM was weaker than that before the chemotherapy.There was significant difference in the positive ratio of PCNA,p53,Bcl-2 and CD44v6 be-tween retreated tumor and tumor.Conclusion:After neoadjuvant chemotherapy with PYM,a large number of tumor cells died.The amplification and metastasis of tumor were suppressed by neoadjuvant chemotherapy with PYM.

Changing the paradigm：the potential for targeted therapy in laryngeal squamous cell carcinoma

Laryngeal squamous cell carcinoma(LSCC) remains a highly morbid and fatal disease. Historically, it has been a model example for organ preservation and treatment stratification paradigms. Unfortunately, survival for LSCC has stagnated over the past few decades. As the era of next-generation sequencing and personalized treatment for cancer approaches, LSCC may be an ideal disease for consideration of further treatment stratification and personalization. Here, we will discuss the important history of LSCC as a model system for organ preservation, unique and potentially targetable genetic signatures of LSCC, and methods for bringing stratified, personalized treatment strategies to the 21^(st) century.

Relationship between the Expression of CD44v6 and Development,Progress, Invasion and Metastasis of Laryngeal Carcinoma

The expression of CD44v6 and its relationship with the development, progress, invasion and metastasis of laryngeal carcinoma was investigated. The expression and content of CD44v6 mRNA in tissuess were detected by both RT-PCR and FCM which were respectively extracted from normal laryngeal mucosa, leukoplakia of larynx, laryngeal papilloma, polyp of vocal cord, tissues of laryngeal carcinoma, metastatic and nonmetastatic lymph nodes of neck, and tissues close to carcinoma. The outcome of RT-PCR indicated that the expression rate of CD44v6 mRNA involved in tissues of laryngeal carcinoma and metastatic lymph nodes of neck was the highest (90 %-100 %) compared with that of leukoplakia of larynx, laryngeal papilloma, tissues close to carcinoma by 0.5 cm (55.56 %-60.00 %) and that of normal laryngeal mucosa, polyp of vocal cord, nonmetastatic lymph nodes and tissues close to carcinoma by 1.0 cm was the lowest ( 13.33 %-20 %). The result from FCM was highly consistent with that from RT-PCR. It was suggested that CD44v6 was closely related with the development, progress, invasion and metastasis of laryngeal carcinoma. The outcome from the tissues close to carcinoma by different distance could do help to the determination of incisal edge in surgery abstractly.

MISSING DIAGNOSIS OF NECK METASTASES BY ROUTINE DETECTING METHOD IN LARYNGEAL CARCINOMAS

Objective To evaluate the missing diagnosis of neck metastases by routine detecting method (palpation combined with one pathological slide) in laryngeal carcinomas.Methods Sixty-six specimens of neck dissections were collected and observed by routine method, transparent method, and continuous sliding method.Results Totally, 1153 lymph nodes were detected by palpation method and another 1204 lymph nodes were detected by transparent method.The lymph nodes detected by transparent method account for 51.1% of the total, and among them 10 metastases were found, which account for 15.6%(10/64) of metastatic lymph nodes.For those with no metastasis detected by routine method, 50 μm interval continuous sliding method was performed, and 14 tiny metastases were found, which account for 21.9%(14/64) of metastatic lymph nodes.Detecting by routine method, most lymph nodes (95%) were in tumor growth and tumor suffusion stage.The missing diagnosis rate of routine method was 37.5%(24/64).Conclusions When routine method was used to detect lymph nodes in neck specimens, missing diagnosis should be considered to select best therapy.Through transparent method small lymph nodes could be found and it is a valuable method to observe pathological changes of small nodes.Continuous sliding method could find micrometastasis precisely, but the work burden is heavy and it is difficult to be widely used.

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.

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.

Expression and significance of PTEN and PCNA in human laryngeal squamous cell carcinoma

Objective: To elucidate the expression and significance of PTEN and PCNA in human laryngeal squamous cell carcinoma. Methods: Immunochemical method was used to study 60 cases of laryngeal carcinoma,20 cases of normal laryngeal tissues which were closely adjacent to carcinoma and 10 cases of normal laryngeal tissues. Results: It was showed that PTEN gene was expressed in 85% laryngeal carcinoma tissues. The percentage of lymph node metastasis of laryngeal carcinoma which were negative or positive of PTEN protein was 77.8% and 33.3% respectively, and the difference was significance (P<0.05). Conclusion: Expression of PTEN in laryngeal carcinoma was different from that of normal laryngeal tissues. It may play a role but not important in the tumorigenesis and development of laryngeal carcinoma.

The Expression and Clinical Significance of ABCG2, Oct4 and Nanog in Laryngeal Carcinoma

Objective: To investigate the protein expression and clinicopathological characteristics of ABCG2, Oct4, Nanog in laryngeal cancer tissues, and to seek new molecular markers for the diagnosis of laryngeal cancer. Methods: The laryngeal cancer tissues and paracancerous tissues of 87 patients with laryngeal carcinoma diagnosed in the department of otorhinolaryngology and head and neck surgery in our hospital from April 2016 to April 2018 were selected as the subjects. QRT-PCR, Real-time PcR, Western blot and immunohistochemical staining were used to detect the expression of ABCG2, Oct4 and Nanog in (Tumor Tissue) and (Adjacent Tissue) in tumor tissue and paracancerous tissue. Results: The results of RT-PCR showed that the positive rates of ABCG2, Oct4 and Nanog in laryngeal carcinoma tissues were 49.30%, 45.07% and 52.11%, respectively, while those in paracancerous tissues were 22.54%, 21.13% and 15.49%, respectively (P < 0.01). The expression of ABCG2, Oct4 and Nanog in laryngeal carcinoma was correlated with tumor differentiation, depth of invasion, age and sex (P < 0.05), but not with tumor size and TNM stage. Conclusion: The expressions of ABCG2, Oct4 and Nanog in cancer tissues are related to tumor differentiation status, and they can be used as new molecular markers for the diagnosis of laryngeal cancer.

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.

Prognostic value of body mass index before treatment for laryngeal squamous cell carcinoma

Objective: Patients with head and neck cancer often suffer from malnutrition. This study aims to investigate the influence of body mass index(BMI) on the prognosis of laryngeal squamous cell carcinoma(LSCC).Methods: A total of 473 patients with LSCC initially treated at Sun Yat-sen University Cancer Center between January 2005 and July 2009 were retrospectively reviewed. Survival analysis was performed by the Kaplan-Meier method and Cox regression model.Results: Low BMI before treatment was significantly associated with poor overall survival in patients with LSCC(P<0.001). BMI was an independent prognostic factor for patients with LSCC.Conclusion: Leanness before treatment was associated with poor prognosis in patients with LSCC. Good nutritional status is favorable to improve survival in patients with LSCC.