The functional analysis of DEGs
According to the criteria that |logFC| > 2 and adj-P-value < 0.05, There are 490 DEGs, among which there are 179 up-regulated genes, and 311 down-regulated genes. The volcano plot was shown in the Fig S1. Secondly, Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed via R software. It shows that endometria cancer is associated with cell cycle, P53 signal pathway, and focal adhesion in KEGG pathway (Fig 1A). GO includes biological process (BP), cellular component (CC) and molecular function (MF). The GO analysis shows that endometria cancer is related to the extracellular matrix organization, cell-cell junction, and cell adhesion molecular binding (Fig 1B).
The methylated driven genes
It is well noted that there is often inverse correlation between DNA methylation and mRNA levels. Then the methylated driven genes are selected out using the “MethylMix” and “corrplot” package in R software. There are 13 methylated driven genes. They are WT1-AS, CDO1, RP11-469H8.6, TMEM132C, GYPC, TSPYL5, VTCN1, DDR2, HSPB6, KLF9, C8orf88, FAXDC2 and PGR. Among them, the expression of RP11-469H8.6 and VTCN1 are upregulated, and other are downregulated. The methylated and expressed heatmap of above 13 DNA are displayed in the Fig 2A and 2B (|R| > 0.3, P < 0.05).
The survival analysis
Survival analysis was performed with the product‐limit method (Kaplan–Meier method). At same time, the log‐rank test (Mantel‐Cox test) was used to compare the difference in the survival status between the high‐ and low‐risk groups via survminer package. Cox model was applied to build a risk model to get the patient's risk value. the coxGene of the risk are TSPYL5, KLF9, GYPC, VTCN1 and PGR. The patient grouping was based on the median value of the risk score (Fig 3A). Obviously, lower risk possesses the better prognosis and higher risk exhibits the poorer prognosis (Fig 3B). The heatmap of TSPYL5, KLF9, GYPC, VTCN1 and PGR expression is shown in Fig 3C. The expression of PGR, GYPC and VTCN1 are divided into high expression group and low expression group. The survival curve of above 13 methylated driven genes were drew by survival software. Three genes (PGR, VTCN1 and GYPC) are considered statistically significant (P < 0.05). The negative correlation of PGR, VTCN1 and GYPC expression and methylation are displayed in Fig 4A, 4B, 4C. The higher expression group of PGR and GYPC displays better prognosis, while the lower expression group shows poor prognosis (P<0.05) (Fig 4DE). Conversely, the lower expression of VTCN1 gene display better prognosis (Fig 4F).
The expression of PGR, GYPC and VTCN1
The expression of PGR, GYPC and VTCN1 were visualized by beeswarm package. It’s obvious that the expression of PGR and VTCN1 are lower in tumor group than that of in normal group. While VTCN1 expression is higher in tumor group than that of in normal group (Fig 5A, 5B, 5C). The protein expression in The Human Protein Altas (https://www.proteinatlas.org/) further confirms the results (Fig 5D, 5E, 5F).
The immune score
CIBERSORT is a deconvolution algorithm that uses a set of reference gene-expression values (a signature with 547 genes) considered a minimal representation for each cell type. Based on those values, it infers cell type proportions in data from bulk tumor samples with mixed cell types using support vector regression. In the immunity low group, StromalScore, ImmuneScore, and EstimateScore are -1257.90±283.05, -378.62 ± 401.33, and -1545.68 ± 608.60, respectively; In the immunity high group, StromalScore, ImmuneScore, and EstimateScore are -539.79±1241.98, 930.89±583.08, and 462.35±834.37, respectively. The survival curve based on the above immunity score shows that higher ImmuneScore displays better prognosis (P<0.05). While StromalScore and EstimateScore shows no statistical significance (P>0.05).
The mechanism of VTCN1 gene in immune regulation
VTCN1 gene is closely related to immunity. In order to further explore the mechanism of VTCN1 involved in the endometria cancer. The relative proteins of VTCN1 are selected via STRING database. They are B7RP1, BTLA, CD28, CD80, CD86, CTLA4, ICOSL, IL4, IL6, and PDCD1LG2 (Fig 6A). Those genes are mainly involved in Lymphocyte costimulation, regulation of T cell activation, proliferation, B cell activation, immune response-regulating cell surface receptor signaling pathway (Table 1). The percentage of immune cells in normal and tumor tissues are analyzed. The box plot is displayed in Fig 6B according to the StromalScore, ImmuneScore, EstimateScore and Tumorpurity. The box plot of the immunity cell percentage in the two group are analyzed by the ggpubr package. It is obvious that B cell native and T cells CD4 memory resting, and M2 macrophagocyte are lower in tumor group than that of normal group. While Tregs and M1 macrophagocyte are higher in tumor group than that of normal group (P<0.05). Likewise, the higher expression of VTCN1 exhibits positive correlation with T cells CD4 memory resting, while the higher expression of VTCN1 is negatively with T cells CD8 and T cells CD4 memory activated cells (Fig 6C). The correction of T cells CD8 and VTCN1 is shown in the Fig 6D.
Table 1
The GO analysis of VTCN1-reatived genes.
Gene lists
|
GO analysis
|
IL4/IL6
|
Lymphocyte costimulation
|
B7RP1
|
Regulation of T cell activation
|
BTLA
|
Regulation of T cell proliferation
|
CD28
CTLA4
|
Control of immune tolerance by Vasoactive Intestinal peptide
|
CD80
|
Cell adhesion molecules (CAMs)
|
CD86
|
B cell activation
|
ICOSL
PDCD1LG2
|
Immune response-regulating cell Surface Receptor signaling pathway
|