| Objective: Lung Cancer is one of the most common malignant tumors in the world.Nonsmall Cell Lung Cancer(NSCLC)accounts for about 80-85% of lung cancers and is the most important pathological type of lung cancer.In 2018 alone,there were approximately2,093,876 new cases of lung cancer and 1,761,007 deaths worldwide.Despite the continuous improvement of comprehensive treatment methods in recent years,the 5-year overall survival rate of patients is still less than 20%.Therefore,actively searching for new markers and therapeutic targets is essential for the diagnosis and treatment of lung cancer.SYT7(Synaptotagmin 7),which was first found to be an asynchronous calcium sensor for neurotransmitter release at the zebrafish neuromuscular junction,and is highly expressed throughout the central nervous system.It has been confirmed that it is one of the most important vesicle calcium sensors,participating in the exocytosis process of dense nuclear vesicles in the fusion of endocrine cells and lysosomes,and it has been found to have functions such as regulating neurotransmitter release and insulin secretion.The migration of cells is related to the development of many diseases.However,in recent years,the role of SYT7 in tumor research,especially in non-small cell lung cancer,remains unclear.Therefore,based on clinical samples,this study attempts to explore the diagnostic value and prognostic role of SYT7 in non-small cell lung cancer through bioinformatics and molecular biology experiments and to clarify the effect of SYT7 on the biological behaviour of non-small cell lung cancer.Further,explore its potential mechanism of action.Therefore,it provides a theoretical basis for SYT7 as a diagnostic,prognostic marker and drug target for patients with non-small cell lung cancer.Methods: 1.Select gene expression profiles from The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)public databases to analyze and study the difference in SYT7 mRNA expression between non-small cell lung cancer and adjacent cancers.2.Collect the GEO gene expression profiles of 17 non-small cell lung cancers and conduct a meta-analysis to clarify the differential level of their expression.3.Analyze the expression level of SYT7 mRNA in the selected online data set,and draw ROC and s ROC curves to prove the diagnostic value of SYT7 expression level for non-small cell lung cancer.4.Analyze the difference in overall survival of patients between SYT7 high and low expression groups in the combined data set through the Kaplan-Meier website.5.Detect the protein expression level of SYT7 in the cancerous tissues of 153 cases of nonsmall cell lung cancer patients and 50 cases of adjacent tissues by immunohistochemistry to determine the difference in the expression of SYT7 protein between cancer and adjacent cancers.6.Collect the clinicopathological parameters of 153 patients with non-small cell lung cancer and the prognostic information of 97 patients with complete follow-up,and analyze the correlation between SYT7 and the clinical phenotype and prognosis of the patients.Cox univariate analysis and Cox multivariate proportional hazard model were used to analyzing the data of 97 non-small cell lung cancer patients with complete followup information to determine whether SYT7 is an independent predictor of patient prognosis.7.By using siRNA and overexpression plasmids,establish the A549 cell line that knocks down SYT7 and the H1299 cell line that overexpresses SYT7,respectively.8.Verify the efficiency of knockdown and overexpression of SYT7 through qRT-PCR and Western Blot experiments.9.Detect the effect of SYT7 on cell proliferation by MTT method and colony formation experiment in non-small cell lung cancer cell lines.10.Detect the effect of SYT7 on cell migration through wound healing experiments and Transwell experiments in nonsmall cell lung cancer cell lines.11.Use R language database software to calculate SYT7 related genes in the TCGA-LUNG data set,and analyze the biological processes,molecular functions and molecular functions involved in SYT7 through Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.12.The changes in the molecular pathway after knocking down and overexpressing SYT7 were detected by Western Blot.13.Statistical analysis with SPSS 20.0 software: each group of experiments was repeated 3 times.T-test compares differences between groups.P<0.05 indicates that the difference is statistically significant.Results: 1.In the data sets of GSE8569,GSE18842,GSE19188,GSE19804,GSE21933,GSE27262,GSE31210,GSE32863,GSE43458,GSE74706,GSE75037,GSE103512,GSE118370 and TCGA-LUNG,the expression level of SYT7 mRNA in non-small cell lung cancer tissues was significantly higher than that in paracancerous normal lung tissues.2.In the TCGA-LUAD and TCGA-LUSC data sets,the expression level of SYT7 mRNA in stage ?-? was higher than that in the adjacent normal lung tissue.3.The meta-analysis of 17 GEO data sets suggests that the expression of SYT7 mRNA in non-small cell lung cancer tissues is higher than that in normal tissues adjacent to cancer.4.The ROC curve of TCGA-LUNG and the s ROC curve of 17 GEO data sets indicate that the expression of SYT7 mRNA has diagnostic value for non-small cell lung cancer.5.Multiple data sets analyzed by the KM-plotter website found that the overall survival time of patients with high SYT7 expression was significantly shorter than those with low expression.6.The immunohistochemical analysis of 153 patients showed that the expression level of SYT7 in non-small cell lung cancer tissues was significantly higher than that in adjacent tissues.7.According to the level of SYT7 protein in non-small cell lung cancer tissues,patients were divided into the SYT7 high expression group and SYT7 low expression group.The analysis of patient clinical sample data indicated that SYT7 was related to the degree of differentiation and p T stage.8.The analysis of clinical sample data of patients indicated that the overall survival(OS)of patients with high SYT7 expression was significantly shorter than that of patients with low SYT7 expression.The Cox proportional hazards models show that SYT7 was an independent risk factor for patient prognosis.9.Verifying the level of SYT7 expression in non-small cell lung cancer cell lines,qRT-PCR and Western Blot results showed that SYT7 expression was highest in A549 cells and lowest in H1299 cells.10.SYT7 knockdown cell line A549 was established by siRNA transfection.SYT7 overexpression plasmid was used to establish the H1299 cell line transfected with SYT7.The qRT-PCR and Western Blot showed that SYT7 knockdown and overexpression were effective.11.Compared with A549-NC cells,the proliferation and migration ability of A549-Si SYT7 cells were weakened.12.Compared with H1299-Vector cells,the proliferation and migration ability of H1299-SYT7 cells were increased.13.Flow cytometry showed that SYT7 knockdown or overexpression did not affect apoptosis and cell cycle in NSCLC.14.SYT7 co-expressed genes were found in TCGA-LUAD and TCGA-LUSC,and GO analysis showed that it could positively regulate the MAPK pathway,while KEGG enrichment analysis suggested that it might affect non-small cell lung cancer by regulating cell adhesion,NF-Kappa B signalling pathway and c AMP signalling pathway.15.Western blot analysis confirmed that knocking out SYT7 could reduce the phosphorylation level of Erk1/2 and p38,but would not change the expression of Erk1/2 and p38.Overexpression of SYT7 promoted the phosphorylation level of Erk1/2and p38 but did not affect the expression of Erk1/2 and p38.Conclusions: 1.SYT7 is highly expressed in non-small cell lung cancer tissues,and its expression has diagnostic value for non-small cell lung cancer.2.SYT7 is related to the poor prognosis of non-small cell lung cancer.3.SYT7 promotes the proliferation and migration of non-small cell lung cancer.4.SYT7 has no significant effect on the apoptosis and cell cycle of non-small cell lung cancer.5.In non-small cell lung cancer,SYT7 can participate in the regulation of the MAPK pathway by affecting Erk1/2 and p38. |
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