3 . 1 | Identification of differential genes in IR and EC
In the GSE63992 data set, we got 594 up-regulated and 526 down-regulated genes to be identified,which are compared with IR patients and normal control group(Figure 1a,c). And in the dataset GSE106191, 3111 up-regulated and 2590 down-regulated genes were obtained in EC group compared with normal control group(Figure 1b ,d). As shown in Figure 1e and Table 1,there were included 81 intersectional upregulated genes and 67 intersectional downregulated genes in IR and EC.
3 . 2 | The gene ontology analysis and pathway of differential genes
We used David online tool to analysis the DEGs in detail,the conclusions of GO analysis indicated that the up-regulated gene changes in biological processes (BP) included significant increasement of several metabolic processes (regulation of DNA-binding transcription factor activity, pattern recognition receptor signaling pathway etc.),as the down-regulated genes were predominantly concentrated in response to glucose,regulation of myeloid leukocyte differentiation and endocytosis(Figure 2a. and Table 2).The molecular function (MF) enrichment analysis indicated the function of the genes mainly concentrated on significant enrichment of binding-related function (protein binding, transcription factor activity binding, and transcription factor binding),as shown in Figure 2b. and Table 2. The changes of cellular component (CC) included significant enrichment of genes related to the cytoplasm and nucleoplasm(Figure 2c. and Table 2).And the changes in KEGG pathway were significantly enriched in NF-kappa B signaling pathway , small cell lung cancer and Epstein-Barr virus infection(Table 3 and Figure 2d).
3 . 3 | Comprehensive analysis of PPI Network
According to the information on GeneMANIA database.There were 148 DEGs to be found , including 81 up- and 67 down-regulated genes in the overall number of 6821 genes , and the PPI network was built with 1364 edges（Figure 3a）. We used the Metascape (http://metascape.org),which is a gratuitous online tool for gene annotation and analysis,to further analyze the PPI network, and selected the top three most significant modules(Figure 3b-d).We also carefully studied the function annotation of the genes in the modules, separately. According to the results of enrichment analysis, the gene functions of these three modules were mainly related to the cell cycle process,actin cytoskeleton organization,ligand binding and protein polyubiquitination(Figure 3e).
Cytoscape is an open source software platform for inquiring about complex networks and assembling these with any type of attribute data.Cytoscape was used to find hub genes.The default limits were as follows: a degree cut-off ≥2, node score cutoff ≥0.2, K-score ≥2 and max depth of 100 for selecting hub genes.Twenty-five hub genes related to EC and insulin resistance were analyzed by multiple algorithms (including EcCentricity,Closeness,Radiality and MNC),and there were used for further analysis.The hub genes were PTEN, YY1, KLF5, RARA, ING3, HES1, BRD4, OGT, E2F2, IGSF3, FBXW7, LYN, PIK3C3, TRAF3, RAD21, PTPN1, PIK3C2B, DDX58, GJB3, TRO, NEURL2, MYL12A, IRAK2, TGFBRAP1, RYBP,respectively.For the connection between EC and IR, the results were revealed in Table 4.
3 . 4 | Expression difference of hub genes in EC
We compared the expression difference of the twenty-five hub genes between EC and normal tissues by using the UALCAN dataset.We discovered that the expression levels of KLF5,BRD4,E2F2,LYN, HES1,YY1,IGSF3,TRAF3and GJB3 were much higher in EC tissues than in normal tissues, and the expression levels of TRO, NEURL2, PTEN, RARA, RYBP, OGT, FBXW7, PIK3C3, PIK3C2B, ING3 and DDX58 were clearly lower in EC tissues than in normal tissues,whereas the expression levels of MYL12A, RAD21,TGFBRAP1,IRAK2and PTPN1 were not significantly different between EC and normal tissues,as shown in Figure 4.
3 . 5 | The histological analysis of hub genes
We compared the twenty-five hub genes between EC and healthy tissues by using the HPA dataset to analysis the expression,Figure 5.The results were the same as UALCAN, the expression levels of KLF5,BRD4,E2F2,LYN,YY1,IGSF3 and TRAF3 were higher in EC tissues than in regular tissues,and the expression levels of TRO, NEURL2, PTEN, RYBP, OGT, FBXW7, PIK3C3, PIK3C2B, ING3 and DDX58 were lower in EC tissues than in normal tissues,whereas the expression levels of MYL12A, RAD21,TGFBRAP1,IRAK2and PTPN1 were not much different between EC and normal tissues.But we did not find RARA,GJB3 and HES1in HPA.
3 . 6 | Survival analysis of the hub genes based on the TCGA dataset
Further analysis was conducted on the above the hub genes,which had expression difference. The Ualcan web tool showed the survival curve of these genes,and the survival curve showed that the survival time of OGT,IGSF3,TRO,NEURL2and PIK3C2B is significantly strongly related to EC (P < 0.05), as shown in Figure 6.And we think about genes, which had survival significant,as candidate genes.
3 . 7 | Genetic alteration analysis in patients with EC
We analyzed the genetic alterations of OGT, IGSF3,TRO,NEURL2 and PIK3C2B in EC by using cBioPortal online tool. A total of 147 patients from four datasets of endometrium cancer were carefully studied. Among the four datasets analyzed, the gene set pathway was changed in 237 (24.6%) of the queried samples, and alterations ranging from 25.52% (135/529) to 13.42% (73/544) were found for the gene sets submitted for analysis(Figure 7a).The percentages of genetic alterations in the five hub genes for EC varied from 3% to 10% for individual genes (OGT, 5%; IGSF3, 4%; TRO, 7%;NEURL2,3% and PIK3C2B, 10%).For OGT, most of the changes are missense mutation, IGSF3 is missense mutation and amplification, TRO is missense mutation, truncation mutation, amplification and deep deletion,NEURL2 is mainly amplification, and PIK3C2B is missense mutation and amplification(Figure 7b). The results of Kaplan–Meier plotter and log-rank test pointed to no significant difference in overall survival (OS) and disease-free survival (DFS) between the cases with changes in one of the query genes and those without alterations in any query genes (P-values, 0.444 and 0.215, respectively; Figure 7c,d)
3 . 8 | Analysis of Tumor-Infiltrating Immune Cells using TIMER tool
Comprehensive analysis of the relationship between the expression of five hub genes and tumor-infiltrating Immune using TIMER.The results showed that OGT, IGSF3,TRO,NEURL2 and PIK3C2B are related to the infiltration of seven kinds of immune cells in endometrial carcinoma.OGT is related to the infiltration of CD8+ T cells and neutrophils(Figure 8a), IGSF3 is related to the infiltration of B cells and CD4+ T cells(Figure 8b),TRO is related to the infiltration of CD8+ T cells and CD4+ T cells(Figure 8c),NEURL2 is related to the infiltration of CD8+ T cells(Figure 8d),and PIK3C2B is related to the infiltration of CD4+ T cells, neutrophils and dendritic cells(Figure 8e).