The Methylation Level Of SHISA3 In The Laryngeal Cancer And Its Clinical Significant Analysis

ObjectiveLaryngeal cancer is one of the most common malignancy of head and neck,the global incidence of laryngeal cancer increases in the recent years.The progression of laryngeal cancer is a multistep process involving intricate interactions between multiple factors,including genetic susceptibility,epigenetic modification and environmental exposure.As an important epigenetic modification,DNA methylation is one of the most common mechanism,which has been shown to associate with numerous cancers.SHISA3 is a novel candidate tumor suppress gene and the hypermethylation of SHISA3 promoter region is responsible for its down-regulation in lung cancer and colorectal cancer.However,the mechanism of SHISA3 in the laryngeal cancer is still unclear.Therefore,in current study,we explore the association of SHISA3 methylation level with laryngeal cancer and its clinical value of diagnosis and prognosis for laryngeal cancer.Methods1.The methylation level and expression of SHISA3 in head and neck squamous cell carcinoma(HNSCC)patients from The Cancer Genome Atlas(TCGA)database were analyzed through the online analysis tool MEXPRESS(http://mexpress.be/).2.A total of 93 paired laryngeal cancer tissue samples and adjacent normal tissue samples,as well as their clinical data,were collected.3.The methylation specific polymerase chain reaction(MSP)was applied to determine the methylation status of SHISA3 promoter in 93 paired laryngeal cancer and adjacent normal tissues.4.The quantitative real-time PCR(qRT-PCR)was performed to measure the expression of SHISA3 in 35 paired laryngeal cancer and adjacent normal tissues.5.The regulatory function of the SHISA3 promoter fragment was analyzed using luciferase reporter assay.6.The chi-squared test or Fisher's exact test was performed to evaluate the differences of SHISA3 promoter methylation frequency between different groups.The comparison of the SHISA3 expression level was analyzed using the paired Student's t-tests.The diagnostic value of SHISA3 was assessed using the receiver operating characteristic(ROC)curve and the area under the curve(AUC).Log-rank tests and multivariate Cox proportional hazard models were used to test the prognostic value of SHISA3 methylation for laryngeal cancer patients.Results1.Based on the results of MEXPRESS,the TCGA data showed elevated methylation level of SHISA3 promoter in HNSCC and inverse relationship between SHISA3 promoter methylation level and its expression.2.The MSP results reveal that there is a significant increase in SHISA3 methylation in laryngeal cancer tissues compared with corresponding non-tumor tissues(P = 4.58E-12).3.The further analysis indicate that SHISA3 promoter methylation is more common events in poor differentiation(P = 0.02),lymph node metastasis(P = 0.03),and advanced clinical stages(P = 0.02),as well as advanced tumor invasion(P < 0.05)laryngeal cancer patients.4.Using qRT-PCR in 35 paired laryngeal cancer samples,it was found that the expression of SHISA3 in laryngeal cancer tissues was significantly downregulated compared with adjacent normal tissues(P = 3.84E-05).In addition,the analysis showed that the expression of SHISA3 was significantly lower in the methylated patients compared with the unmethylated patients(P = 1.67E-03).5.The reporter gene activity assay revealed that the methylated SHISA3 promoter fragment was able to regulate gene expression.6.A ROC curve was plotted to evaluate the diagnostic value of SHISA3 promoter methylation and the area under the ROC curve is 0.91(P < 0.01).The survival curves demonstrated that hypermethylated SHISA3 is significantly associated with poor outcome of laryngeal cancer(log-rank P = 0.024).A multivariate Cox proportional hazard analysis confirmed a significantly poor prognosis in laryngeal cancer patients with SHISA3 methylation(hazard ratio(HR),2.71;95 % confidence interval,1.024–7.177;P = 0.047).ConclusionThe hypermethylation of SHISA3 promoter is a common event in laryngeal cancer,contributing to its transcriptional inactivation,and may be involved in the invasion,progression and metastasis of laryngeal cancer,could as a potential early diagnostic and prognostic biomarker for the laryngeal cancer.