| Objective: Thyroid carcinoma is the most common endocrine malignancy,and its incidence is increasing worldwide.Although the vast majority of papillary thyroid cancer(PTC)patients have a favorable prognosis after conventional treatments,local recurrence and distant metastasis of advanced PTCs still hamper the survival and clinical management in certain patients.Numerous studies have confirmed that Immunotherapy achieves great success and remarkable breakthroughs in some advanced cancers.This study aimed to construct an immune-related prognostic signature to identify potential therapeutic targets and investigate the tumor immune microenvironment that could effectively predict the survival and prognosis for thyroid carcinoma.Additionally,to explore the expression of SEMA6 B in thyroid carcinoma and non-tumorous tissues,and the prognostic value of SEMA6 B expression.To investigate the potential effects of SEMA6 B on proliferation,migration and invasion of thyroid carcinoma cells,so that to provide new ideas and perspective for individualized and targeted treatment of thyroid carcinoma.Methods: 1.Based on The Cancer Genome Atlas(TCGA)database,we downloaded and integrated gene expression data and clinical data from thyroid carcinoma patients with complete clinical information.Differentially expressed genes(DEGs)were identified and calculated using the “limma” package in R language,and the differentially expressed matrixes of immune-related genes(IRGs)were extracted.The biological functions of IRGs of thyroid carcinoma were explored by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)functional enrichment analysis.Combined with clinical follow-up information,we performed univariate Cox regression analysis to screen out IRGs related to the prognosis of thyroid carcinoma.Subsequently,a least absolute shrinkage and selection operator(LASSO)Cox regression analysis with the “glmnet” package was used to avoid overfitting.Finally,the immune-related prognostic risk signature was constructed through multivariate Cox regression analysis.Kaplan-Meier survival curve and Receiver operating characteristic curve(ROC)were performed to evaluate the accuracy and reliability of the risk signature.Additionally,univariate and multivariate Cox regression analyses on the prognostic signature and multiple clinical information were utilized to determine whether the risk signature could act an independent predictor for thyroid carcinoma.Furthermore,the correlation between the immune-related prognostic signature and clinicopathological characteristics,and tumor-infiltrating immune cells in the immune microenvironment(TME),and the response of potential immunotherapeutic were also discussed.At last,we verified the expression levels of the prognostic risk genes in 98 pairs of tumorous and non-tumorous tissues using quantitative Real-time PCR(q RTPCR)analysis.2.Biological function study of SEMA6 B.(1)Expression of SEMA6 B was investigated by q RT-PCR,immunohistochemistry(IHC)and Western Blot in PTC tissues and adjacent tissues,as well as PTC cell lines and normal cell lines.Overall survival was evaluated to elucidate the prognostic value of SEMA6 B.(2)The expression of SEMA6 B was inhibited through lentiviral transfection,and the interference efficiency was verified by q RT-PCR and Western Blot technology.(3)The scratch assay,cell counting kit-8(CCK8)and Transwell assay were used to evaluate the migration,proliferation and invasion of thyroid carcinoma cells after downregulation of SEMA6 B.Results: 1.Transcriptome RNA-sequencing data of thyroid carcinoma samples with complete clinical information were downloaded and integrated from TCGA database.According to the adjusted p value of < 0.05 and a |log2(fold change)of | > 1,a total of2413 DEGs were identified based on the RNA expression profile data of thyroid carcinoma,among which 1559 were up-regulated and 854 were down-regulated,and there were 262 DE-IRGs.GO functional enrichment analysis showed that the enriched biological process(BP)mainly included cell chemotaxis and the humoral immune response;the most enriched cellular component(CC)was the external side of the plasma membrane;and receptor–ligand activity and signaling receptor activation were significantly enriched in terms of molecular function(MF).The most significant pathway that they may participate in was cytokine-cytokine receptor interactions.Univariate Cox regression analysis showed that 41 DE-IRGs were closely associated with overall survival(P < 0.05).LASSO Cox regression analysis was performed on 41DE-IRGs.According to λ value,combined with multivariate Cox regression analysis,a total of 8 IRGs(HSPA6,PPBP,S100A11,AZU1,SEMA6 B,VGF,IL20 RA,and FYN)were finally screened out to construct an immune-related prognostic signature,and the risk score each sample was calculated.All patients were separated into high-and lowrisk subgroups according to median risk score.Kaplan-Meier survival curve indicated that the overall survival of patients in high-risk groups was significantly worse than that in low-risk groups.The areas under the ROC curve(AUC)revealed a better accuracy and reliability of the risk signature.Univariate and multivariate Cox regression analyses showed that the risk score could be regarded as an independent prognostic factor.Clinical correlation analysis revealed that the risk score was significantly associated with age and lymph node metastasis.Furthermore,the relative proportions of 21 types of tumor-infiltrating immune cells of the prognostic signature were evaluated.The risk score was positively correlated with monocytes and dendritic cells infiltrated in the TME.Nevertheless,plasma cells were negatively correlated with risk score.Immunophenoscore(IPS)analysis showed that IPS was significantly higher in low-risk group.The expression levels of common immune checkpoint proteins(PD-L1,PD-L2,TIGIT,TIM-3,and CTLA4)were significantly higher in low-risk group.The results of the q RT-PCR showed that the expression levels of 8 prognostic genes in 98 pairs of tumorous and non-tumorous tissues showed significant differences,and the expression levels were consistent with the results of bioinformatic analysis.2.Analysis of q RTPCR,IHC and Western Blot results showed that compared with normal thyroid carcinoma tissues and cells,SEMA6 B expression was significantly up-regulated in thyroid carcinoma tissues and cell lines.Survival analysis suggested that the overall survival of the SEMA6 B high subgroup was significantly shorter than SEMA6 B low subgroup(P < 0.05).The proliferation,migration and invasion of thyroid carcinoma cells can be inhibited when expression level of SEMA6 B was decreased.Conclusion: 1.We screened out 8 immune genes with clinical significance to construct immune-related prognostic signature for thyroid carcinoma.These genes could be used promising prognostic biomarkers in patients with thyroid carcinoma,which can effectively predict the prognosis of patients to a certain extent.This immune signature might provide a new perspective for future immunotherapies and personalized clinical therapeutic strategy.2.SEMA6 B expression is up-regulated in thyroid carcinoma tissues and cells and the high expression of SEMA6 B indicates a poor prognosis.Down-regulation of SEMA6 B expression inhibits proliferation,migration and invasion of PTC cells,and could be used as a potential therapeutic target for PTC. |
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