| The proportion of lung adenocarcinoma(LUAD)in global cancer is gradually increasing,and the survival rate of cancer(22%)in the past five years is not optimistic.Therefore,it is necessary to explore molecular markers that can not only accurately and stably predict LUAD,but also become potential therapeutic targets.Ubiquitination and de-ubiquitination(DUB)are widely involved in various processes of protein modification and regulation,and abnormal regulation disorder leads to tumorigenesis.With the increasing recognition of the key importance of ubiquitination in cell processes,the role of DUB in health and disease is becoming a new focus of scientific research.The latest progress in this field has revealed more and more DUB substrates.The discovery of more specific biomarkers related to de-ubiquitination may provide new clues for the diagnosis and targeted treatment of LUAD.Download the LUAD data in the TCGA and GEO databases.R software package SVA is used to eliminate batch effects in GEO data sets.The limma software package detects the DEGs in the GEO data set,and the edger software package detects the DEGs in the TCGA data set.The R software package cluster Profiler is used for enrichment analysis.The Consensus Cluster Plus software package is used for cluster analysis.The experimental interaction and prediction information of DEGs protein was obtained by searching the interaction gene retrieval tool STRING.Cytoscape constructed the specific protein interaction(PPI)network of DEGs,and the cytohubba plug-in was used to screen the target gene.GEPIA database is used to verify the expression of target genes,and Kaplan-Meier database is used to explore the prognostic value of target genes.χ~2 Test is used to test the correlation between the target gene and clinical parameters,and further verify in UALCAN.The tissue microarray(TMAs)composed of 150 patients with lung adenocarcinoma was prepared,and the expression of UCHL1 protein was detected by immunohistochemistry(IHC).The human protein atlas(HPA)database is used to analyze the expression level of UCHL1 in various tumor cell lines and the immunofluorescence expression profile of UCHL1 in A549 cell lines.CCLE database is used to explore the expression data of UCHL1 in lung adenocarcinoma cells.LDN-57444 was used to inhibit the expression of UCHL1 gene in A549 cells,and the effect of gene inhibition was detected by immunocytochemistry and immunoblotting.LDN-57444 inhibited the change of cell function caused by UCHL1,which was verified by scratch test and Transwell test.The batch effect of GEO dataset is effectively eliminated through SVA function,and the final dataset is obtained(LUAD organization: corresponding normal adjacent organization=199:190).A total of 2471 DEGs were screened from the GEO dataset,8704 DEGs were screened from the TCGA-LUAD dataset,and 24 overlapping DEGs were related to de-ubiquitination.De-ubiquitination related DEGs mainly play the functions related to protein de-ubiquitination,such as protein hydrolysis,protein modification,protein metabolism,and participate in viral carcinogenesis,cell cycle,cell aging,necrosis,NOD-like receptor signal pathway,and IL-17 signal pathway.LUAD patients were divided into three subtypes by consensus clustering: cluster 1(N=168),cluster 2(N=155),and cluster 3(N=192).Cluster 2 and cluster 3 differ greatly in gene expression and prognosis.Compared with cluster 2,the expression of de-ubiquitination related DEGs in cluster 3 patients is relatively high,and the prognosis of LUAD patients in cluster 3 group is poor.A total of 24de-ubiquitization-related DEGs were imported into the PPI network,including 21 nodes and 46 edges.Determine the target genes(UCHL1,PSMD11,PSME4,NFKBIA)from the PPI network through the cytohubba plug-in.The expression of UCHL1 was up-regulated in LUAD,and the prognosis of patients with high expression was poor(P=2.8e-05),which was related to the gender and tumor stage of patients.The expression of PSMD11 was up-regulated in LUAD,and the prognosis of patients with high expression was better(P=1.7e-10).The analysis of the difference in expression of PSME4 in LUAD,prognostic correlation(P=0.22),and clinical correlation was significantly insufficient.NFKBIA is down-regulated in LUAD,and the prognosis of patients with high expression is poor(P=0.00058),which is related to gender and tumor stage.The highly expressed UCHL1 shows good clinical diagnostic value for LUAD and can be used for subsequent tissue and functional verification.UCHL1 in TMAs showed a higher level of UCHL1 protein in lung adenocarcinoma tissue,which was related to the age of LUAD patients and tumor size.Interestingly,there was a significant positive correlation between UCHL1 and MKI67 expression.The expression analysis of UCHL1 in various tumor cell lines showed that the expression of UCHL1 in lung cancer cell lines ranked third.In lung adenocarcinoma cell lines,UCHL1 was more significantly expressed in A549 cells.The inhibition of UCHL1 by LDN-57444 leads to the reduction of cell migration and invasion.After UCHL1 inhibition,A549 cells showed a higher probability of apoptosis.This study combined TCGA and GEO databases to analyze the de-ubiquitination related DEGs in LUAD.Cluster subtype analysis revealed the relationship between the expression of de-ubiquitination related DEGs and the prognosis of LUAD.According to further screening,the key gene UCHL1 was found to have significant predictive effect in the diagnosis,prognosis and clinical characteristics of LUAD.In addition,functional experiments revealed that UCHL1 can enhance the migration and invasion ability of A549 cells and inhibit cell apoptosis.UCHL1 can be used as a sensitive indicator for the early diagnosis of lung adenocarcinoma,providing a possible target for the development of targeted drugs for lung adenocarcinoma.Figure 18 Table 3 Reference 161... |
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