| Background:Lung cancer is the most common malignant and fatal tumor in the world,which seriously threatens human health and quality of life.The most common type of lung cancer is non-small cell lung cancer(NSCLC),and the main types of NSCLC are lung adenocarcinoma(LUAD)and lung squamous cell carcinomalung(LUSC).Despite the improvement of treatment strategies,the prognosis of patients with NSCLC is still poor,and the lack of timely diagnosis and effective treatment are the main reasons for the poor prognosis of NSCLC.Therefore,the study on the mechanism of the occurrence,development and metastasis of NSCLC at the molecular level and the discovery of new key genes and biomarkers can provide an important early-stage basis for the early detection and precise treatment of patients with NSCLC.Based on this,our research group found that glycoprotein 6A(GPM6A)and intelectin-2(ITLN2)may be potential important biomarkers and therapeutic targets through bioinformatics screening.GPM6 A is a member of the protein lipoprotein(PLP)family,located on chromosome 4q34.2,involved in the regulation of stress,mood,appetite,immune response and other functions.The role of GPM6 A in tumor is still controversial.A study using gene expression database analysis found that GPM6 A is underexpressed in lung cancer,and down-regulated expression is one of the adverse factors affecting the prognosis of patients,suggesting that GPM6 A may be a potential tumor suppressor gene in lung cancer.ITLN2,a member of the endagglutinin family,is located on chromosome 1q22-23.5.Currently,most studies on ITLN2 focus on immunity,and ITLN2 expression is up-regulated after a variety of bacterial infections.No studies on the correlation of ITLN2 in NSCLC have been reported.Whether GPM6 A and ITLN2 participate in the occurrence,development and metastasis of NSCLC,and the possible molecular mechanism remains unclear.Research purposes: To determine the clinical value and significance of GPM6 A and ITLN2 in NSCLC;To clarify the main functions of GPM6 A and ITLN2 in NSCLC;To elucidate the molecular mechanism of GPM6 A and ITLN2 in NSCLC.Methods:1.TIMER2.0 database and UALCAN database were used to analyze the expression of GPM6 A and ITLN2 in pan-cancer.UCSC XENA database was used to download TCGA cohort related clinical data,GPM6 A and ITLN2 expression data,gene promoter region methylation data and gene copy number variation data.2.Graph Pad Prism 8.0 and SPSS25.0 were used to analyze the mRNA expression of GPM6 A and ITLN2 between NSCLC tumor tissues and normal tissues,and the correlation between GPM6 A and ITLN2 expression and clinical indicators of patients.The expression and clinical correlation of GPM6 A and ITLN2 were verified by GEO data sets.In addition,the expressions of GPM6 A and ITLN2 in normal lung cells and NSCLC cells were detected by qRT-PCR.The promoter methylation data and gene copy number variation data were used to analyze the causes of its expression changes.3.Survival analysis was performed using Kaplan-Meier Plotter,GEPIA 2.0 and Onco Lnc online websites to evaluate the prognostic value of GPM6 A and ITLN2.4.The diagnostic value of GPM6 A and ITLN2 for NSCLC,LUAD and LUSC was evaluated by calculating the area under the receiver operating characteristic curve(ROC).5.Quantitative analysis of GPM6 A protein by immunohistochemical staining(IHC)was performed by constructing tissue chip to further verify the clinical correlation,prognosis and diagnostic values of GPM6 A expression at the protein level.6.ESTIMATE database was used to analyze the correlation between GPM6 A and ITLN2 expression and tumor microenvironment related indicators.The data of 24 kinds of immune cell infiltration in NSCLC patients were downloaded from TCGA database,and the correlation between GPM6 A and ITLN2 expression and 24 kinds of immune cell infiltration was analyzed.The relationship between expression levels of GPM6 A and ITLN2 and immune cell infiltration was further verified by TIMER2.0 database.7.By constructing differential expression vectors,NSCLC cells were transfected,and the differential expression of GPM6 A and ITLN2 was verified by qRT-PCR and WB experiments.CCK8,colony formation,flow cytometry and transwell assays were used to analyze the effects of GPM6 A and ITLN2 on the cell proliferation,cycle,apoptosis,migration and invasion of NSCLC cells in vitro.8.The co-expressing genes of GPM6 A and ITLN2 in LUAD and LUSC were obtained from Linkedomics database.Function annotations were input into the DAVID database for GO(Gene ontology)and KEGG(Kyoto encyclopedia of genes and genomes).STRING and Gene MANIA databases were used to construct the GPM6 A and ITLN2protein-protein interaction(PPI)networks,and functional annotation of GO and KEGG were performed.To find the most likely downstream target molecules and signaling pathways for its function.9.In the cell model of differential expression,qRT-PCR,WB and other experiments were used to detect the downstream key molecules and signaling pathways.On this basis,the differentially expressed cell model was treated with agonists or inhibitors of related molecules,and the recovery of cell function was detected by CCK8,apoptosis,and transwell experiments.Results:1.The expression characteristics,role and possible mechanism of GPM6 A in NSCLC(1)The expression of GPM6 A was down-regulated in NSCLC,LUAD and LUSC(P<0.05),and was related to clinical stage and Tumor size(P<0.05).(2)Kaplan-Meier survival curve showed that patients with high GPM6 A expression in NSCLC had better prognosis than those with low GPM6 A expression(Logrank P=3.3e-6),which was correlated with clinical stage.In LUAD,patients with high GPM6 A expression had better OS than those with low expression(Logrank P=1.9e-7),which was correlated with clinical stage,and this result was verified by immunohistochemical staining results.However,in LUSC,patients with high GPM6 A expression showed no significant difference in prognosis compared with patients with low GPM6 A expression(Logrank P=0.21),and there was no significant correlation with clinical stage.(3)ROC curve analysis showed that the area under the curve(AUC)of GPM6 A expression in NSCLC,LUAD and LUSC was greater than 0.98,had very high sensitivity and specificity.(4)The CNV of GPM6 A in NSCLC was mainly deletion.In LUSC,the percentage of GPM6 A copy number deletion was as high as 67.27%.The gene expression of GPM6 A copy number deletion patients was lower(P<0.05),and it was positively correlated with the copy number(P<0.05).However,there was no correlation between GPM6 A copy number and its expression in LUAD(P>0.05).In LUAD patients,the degree of GPM6 A promoter methylation was higher than that in LUSC patients(P<0.05).The expression of GPM6 A was negatively correlated with the β value of promoter methylation,and patients with hypermethylation had lower expression than those with hypomethylation(P<0.05).There was no significant correlation between GPM6 A expression and promoter methylation in LUSC patients(P>0.05).(5)In NSCLC,LUAD and LUSC,GPM6 A expression was significantly correlated with stromal score,immunoscore and ESTIMATE score(P<0.05).Next,TCGA cohort data were used to analyze the correlation between GPM6 A and 24 immune cells.In NSCLC,GPM6 A expression level was correlated with 22 types of immune cells(P<0.05).In LUAD,expression level of GPM6 A was correlated with 19 types of immune cells(P<0.05).In LUSC,the expression level of GPM6 A was correlated with 22 types of immune cells(P<0.05).TIMER database analysis showed that the infiltration levels of B cells,CD4+T cells,CD8+T cells,neutrophils,macrophages and dendritic cells were positively correlated with the expression of GPM6 A in LUAD and LUSC(P<0.05).(6)Colony formation assay,CCK8 assay and flow cytometry showed that overexpression of GPM6 A could block NSCLC cells in S phase,inhibit cell growth and proliferation,and induce cell apoptosis(P<0.05).Transwell experiment showed that overexpression of GPM6 A could reduce the number of NSCLC cell migration and invasion(P<0.05).(7)In LUAD and LUSC,GO enrichment analysis showed that GPM6 A was involved in biological processes such as signal transduction,immune response and positive regulation of apoptosis process.KEGG enrichment analysis showed that GPM6 A was involved in RAS signaling pathway in both isoforms.Further studies showed that the key molecule of RAS downstream ERK1/2 was down-regulated after GPM6 A overexpression,and the phosphorylation level was significantly decreased(P<0.05),suggesting that GPM6 A may be involved in the regulation of ERK1/2 and its phosphorylation.Treatment of GPM6 A overexpression cells with the agonist of ERK1/2(Bortezomib)showed that the inhibitory effect of GPM6 A overexpression on cell proliferation,apoptosis,migration and metastasis was alleviated(P<0.05).These results suggest that phosphorylation of ERK1/2 may play an important role in the inhibition of tumor growth and metastasis by GPM6 A.2.The expression characteristics,role and possible mechanism of ITLN2 in NSCLC(1)The expression of ITLN2 was down-regulated in NSCLC,LUAD and LUSC(P<0.05),and was correlated with Tumor size(P<0.05).(2)Kaplan-Meier survival curves showed that higher ITLN2 expression was associated with better prognosis in LUAD patients,but not in LUSC patients,suggesting that ITLN2 may be a prognostic marker in LUAD patients.(3)ROC curve analysis showed that ITLN2 had a good diagnostic value for NSCLC,LUAD and LUSC(AUC >0.98),with very high sensitivity and specificity.(4)In TCGA database,ITLN2 expression was correlated with the infiltration of 9types of immune cells in NSCLC(P<0.05),16 types of immune cells in LUAD(P<0.05),9 types of immune cell infiltration in LUSC.TIMER database analysis showed that the expression level of ITLN2 was positively correlated with B cell,CD4+T cell,CD8+T cell and macrophage infiltration in NSCLC(P<0.05).The expression of ITLN2 was positively correlated with B cell,CD4+T cell,macrophage and dendritic cell infiltration in LUAD(P<0.05).In LUSC,the expression of ITLN2 was positively correlated with the infiltration of CD8+T cells,neutrophils and macrophages(P<0.05).(5)Functional experiments showed that ITLN2 inhibited cell proliferation and cell cycle arrest in G1 phase,inducing cell apoptosis in NSCLC cells(P<0.05).Overexpression of ITLN2 reduced the number of migration and invasion of NSCLC cells(P<0.05).(6)GO and KEGG enrichment analysis showed that ITLN2 was mainly involved in biological processes such as cell adhesion,angiogenesis,signal transduction and cell surface receptor signaling pathways in LUAD,and was related to signaling pathways such as complement and coagulation cascade,PPAR,and calcium ions.In LUSC,ITLN2 is mainly involved in biological processes such as signal transduction,upper cutaneous branch in lung morphology,cell communication and vasoconstriction regulation,and is related to RAS signaling pathway,PPAR signaling pathway and PI3K-AKT signaling pathway.Further analysis showed that PPARγ and PPARα were positively correlated with the expression level of ITLN2 in NSCLC(P<0.05),and PPARβ/δ was negatively correlated with the expression level of ITLN2 in NSCLC(P<0.05).In LUAD,PPARγwas positively correlated with ITLN2 expression levels(P<0.05).In LUSC,PPARγ was positively correlated with ITLN2 expression level(P<0.05),and PPARβ/δ was negatively correlated with ITLN2 expression level(P<0.05).It is suggested that the role of ITLN2 in NSCLC may be related to PPAR signaling pathway.(7)PPI analysis showed that ITLN2 interacted with ITLN1,ENDOG,ANGPTL3 and RETNLB.Enrichment analysis of these interacting proteins showed that ITLN2 interacting proteins were involved in protein ubiquitination,apoptosis,negative regulation of Rho protein signaling and transduction biological processes.It is mainly involved in apoptosis,cancer signaling pathway,P53 and other signaling pathways.ITLN2 may be mainly involved in the process of tumor cell apoptosis in NSCLC.Further analysis showed that the expression level of P53 was positively correlated with the expression level of ITLN2(P<0.05),and the expression levels of P53 downstream molecules P21 and BAX were also positively correlated with the expression level of ITLN2(P<0.05).These results suggest that ITLN2 may function by interacting with P53 signaling pathway proteins.Conclusion:GPM6A and ITLN2 are important tumor suppressor genes in NSCLC.GPM6 A plays the antitumor roles by regulating ERK1/2 and its phosphorylation level and affecting the tumor microenvironment.ITLN2 exerts its anticancer function by affecting PPAR and P53 signaling pathways and tumor microenvironment. |
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