Bioinformatics Analyses Of Biomarkers Associated With Prognosis In Stomach Adenocarcinoma

Hundreds of thousands newly confirmed cases of stomach adenocarcinoma(STAD)were reported each year in our country,which severely burdened public health system.Patients with STAD had an extremely poor prognosis because of the lack of prognostic biomarkers,despite the treatment had been improved.This paper built a simplified prognostic model based on bioinformatics analyses and discussed the mechanism and molecular functions of the prognostic model.Furthermore,the clinical samples and cell experiment were used for verification.The main findings are as follows:(1)Excavation of potential prognostic markers of STAD.By analyzing the three STAD datasets in the GEO database,44 DEGs were identified.Among them,5 DEGs which affected the survival of STAD patients were screened out according to survival analysis.COX regression was applied to build a prognostic model independent from age,gender and tumor stage,which could divide patients into high-risk group and lowrisk group.The prognostic model contained two variants,LRFN4 and CTHRC1,which were verified using another dataset.Immunological analysis indicated that the infiltration degree of 6 types of immune cells between the high-and the low-risk groups were different.The two immune checkpoints,TIM-3 and PD-L2,were highly correlated with the risk score.Pathway enrichment analysis showed that multiple signaling pathways such as neuroactive ligand receptor interaction,extracellular matrix(ECM)receptor interaction and hypertrophic cardiomyopathy had large differences between the two groups of patients.These results indicated that LRFN4 and CTHRC1 can be used as potential markers for the clinical prognosis prediction and treatment of patients with STAD.(2)The analysis of potential biological functions of LRFN4.The expression level of LRFN4 was affected by mutation of TTN,SYNE1 and ARID1 A,and was related to tumor genome features including microsatellite instability(MSI)and tumor mutational burden(TMB).LRFN4 had better prognostic value and the significant negative correlation with the immune checkpoint of VTCN1 in female.LRFN4 was related to the infiltration of 3 types of immune cell,such as eosinophils.Correlation analysis revealed that there was a potential regulatory pathway of LRFN4-IL33/TSLP–IL5–eosinophilic between LRFN4 and eosinophils.Pathway enrichment analysis showed that LRFN4 participated in multiple metabolic pathways of tumor growth and proliferation.Coexpression network analysis revealed that LRFN4 might maintain a better prognosis in STAD patients through promoting the function of cell apoptosis and inhibiting tumor cell invasion and metastasis.(3)The analysis of potential biological functions of CTHRC1.The expression level of CTHRC1 was increased in the late stage of STAD,and its prognostic value was better in young patients.Immune cell infiltration analysis showed that CTHRC1 was related to the infiltration of 7 immune cells and the expression of 5 immune checkpoints.Pathway enrichment analysis revealed that the high expression of CTHRC1 was related to the multiple pathways involved in tumor occurrence,development,metastasis,and invasion.Based on gene co-expression network analysis,the expression level of CTHRC1 in plasma might be a potential diagnostic marker for early diagnosis ofe STAD patients.(4)The results of single-cell sequencing data analysis showed that there was a big difference in cell composition between tumor and normal tissues.Macrophages only existede in tumor tissues,while parietal cells only existed in normal tissues.The expressions of LRFN4 and CTHRC1 had been detected in all five types of cells.The proportion of cells expressing LRFN4 and CTHRC1 in tumor tissues was higher than that in normal tissues,but the expression levels of LRFN4 and CTHRC1 in a single cell in tumor tissues were not different from normal cells.Therefore,the high expression of LRFN4 and CTHRC1 in tumor tissues was not directly related to the increased gene expression level in a single cell,but it might be the increased proportion of cells with LRFN4 and CTHRC1 expression in tumor tissue.In addition,6 sample cases and cell experiment results showed that the expression level of LRFN4 and CTHRC1 in tumor tissues was higher than that in normal tissues,but the expression levels in tumor cells were not higher than that in normal cells.The high expression of LRFN4 and CTHRC1 in patient tumor tissues is because ofe the increased proportion of cells with LRFN4 and CTHRC1 expression in tumor tissues,rather than the increase in expression in a single cell.The analysis results showed that LRFN4 and CTHRC1 can be used to predict the prognosis of STAD patients.These results will be benefit for developing new diagnostic and prognostic biomarkers for STAD,understanding the relevant mechanisms of STAD progression,and provide clues for future research.