Development And Verification Of A Gene Signature Of Head And Neck Squamous Cell Carcinoma Based On MTORC1 Signaling Pathway

Background: Head and neck squamous cell carcinoma(HNSCC)is the most common pathological type of head and neck malignant tumors,accounting for more than 90%.HNSCC is a fatal disease with a low survival rate and high metastasis rate.Looking for an effective risk stratification method and prognostic markers will help strengthen the management of HNSCC disease.The mammalian target of rapamycin complex 1(mTORC1)signaling pathway plays an important role in the occurrence and development of HNSCC,but its related gene signature has not been developed so far.Therefore,we aim to construct a new gene signature based on the mTORC1signaling pathway through bioinformatics analysis,which could predict the prognosis of HNSCC patients and their response to chemotherapy and immunotherapy.Moreover,we aim to explore specific genes related to the progress and prognosis of HNSCC through gene signature,and further investigate their expression level and function.Methods:1.We extracted the gene expression matrix and clinical data of HNSCC patients from The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)databases,and performed gene set enrichment analysis(GSEA)of mTORC1signaling pathway.Genes related to the prognosis of HNSCC in the mTORC1pathway were screened by univariate Cox regression analysis.Then,according to the key genes identified by multivariate Cox regression analysis,a gene signature related to prognosis was established.Kaplan-Meier(K-M)survival analysis and receiver operating characteristic(ROC)curve were used to analyze and evaluate the performance of the gene signature.According to the median risk score,HNSCC patients were divided into high-risk group and low-risk group.Then,we built a nomogram to predict the prognosis of HNSCC patients through independent prognostic analysis of age,sex,histological grade,AJCC stage and risk score,and evaluated it through calibration chart and decision curve analysis(DCA).In addition,we also compared the differences of immune cell infiltration and responses to chemotherapy and immunotherapy between the two risk groups.Finally,we detected the expression level of seven genes by quantitative real-time polymerase chain reaction(qRT-PCR)and immunohistochemistry(IHC).2.We further explored the relationship between SEC11 A and the prognosis of HNSCC and its role in tumor progression.The expression level of SEC11 A in 18 pairs of cancer and adjacent normal tissues was detected by qRT-PCR and Western blot(WB).IHC was performed on the cancer tissue and adjacent normal tissue samples collected from HNSCC patients to evaluate the expression level of SEC11 A in HNSCC patients and its relationship with prognosis.In addition,we used the lentivirus-mediated SEC11 A knockdown cell model in vitro to study the functional role of SEC11 A in the proliferation and progression of HNSCC.The cell proliferation potential was evaluated by colony formation assay and CCK-8(Cell Counting Kit-8)assay,and the cell migration and invasion ability was evaluated by wound healing assay and transwell assay.In order to evaluate the tumorigenicity of HNSCC cells in vivo,we carried out a tumor xenograft assay in nude mice and conducted IHC on subcutaneous tumors.Result:1.GSEA results showed that mTORC1signaling pathway related genes were up-regulated in HNSCC cancer tissue.A total of seven genes(SEC11A,CYB5 B,HPRT1,SLC2A3,SC5 D,CORO1A and PIK3R3)closely related to the prognosis of HNSCC patients were screened out through Cox regression analysis,and a risk score formula(gene signature)based on mTORC1signaling pathway was constructed.In TCGA and GEO databases,HNSCC patients were divided into high-risk group and low-risk group based on the median risk score.K-M survival analysis showed that the overall survival(OS)of patients in the high-risk group was lower than that in the low-risk group.Through univariate and multivariate COX regression analysis,the gene signature was confirmed as an independent prognostic risk factor for HNSCC.2.Through independent prognostic analysis,we constructed a nomogram based on age,AJCC(American Joint Committee on Cancer)stage and risk score to predict the prognosis of HNSCC patients.The calibration curve showed that the nomogram has good accuracy in predicting the 3 and 5 year survival rate of HNSCC patients.DCA shows that the net benefit of nomogram is higher than that of age,AJCC stage and risk score.In addition,there were differences in the distribution of immune infiltrating cells between the high-risk group and the low-risk group.High and low risk groups showed different responses to five chemotherapeutic drugs,with the low risk group being more sensitive to PD-1 inhibitor.3.By conducting qRT-PCR and WB tests on cancer tissues and adjacent normal tissues of HNSCC patients,we found that SEC11 A was significantly overexpressed in cancer tissues.IHC results showed that SEC11 A was mainly located in the cytoplasm,and its expression level was significantly correlated with the prognosis of HNSCC patients(P<0.01).4.Two cell lines with high expression of SEC11A(TU212 and TU686)were screened from seven common HNSCC cell lines,and stable SEC11 A knockdown cell lines was constructed using shRNA lentivirus.qRT-PCR and WB showed that lentivirus knockdown efficiency was high.Colony formation assay and CCK-8 test showed that knockdown of SEC11 A could reduce the proliferation ability of HNSCC cells in vitro;Wound healing assay and transwell assay showed that SEC11 A knockdown could reduce the migration and invasion ability of HNSCC cells in vitro.In addition,the tumor xenograft assay in nude mice showed that the down-regulation of SEC11 A could significantly inhibit the growth of subcutaneous tumors in mice.IHC of mouse tumor tissue also showed that the proliferative ability of tumor cells with knockdown of SEC11 A was decreased.Conclusion: We established and verified a gene signature based on mTORC1signaling pathway,which can be used for risk stratification of HNSCC patients.The nomogram based on gene signature can be used as a new prognostic model for HNSCC.In addition,the gene signature can be used as a marker to predict the sensitivity of HNSCC patients to chemotherapy and immunotherapy.Therefore,the development and verification of the gene signature is conducive to disease management and individualized treatment of HNSCC patients.SEC11 A was overexpressed in HNSCC tumor tissue and was significantly associated with the prognosis of patients.SEC11 A knockdown reduced the proliferation,migration and invasion of HSNCC cells in vitro and inhibited the growth of subcutaneous tumors in vivo.SEC11 A plays a crucial role in the proliferation and progression of HNSCC,and is expected to become a new therapeutic target.