2.1 GTSE1 is substantially expressed in ccRCC
We discovered that GTSE1 was strongly expressed in most malignancies, including ccRCC, using the timer database (Figure 1A). TCGA, GSE53757, and GSE40435 transcriptome data revealed that GTSE1 in ccRCC tissues was considerably greater than in nearby normal tissues (Figure 1B-D). Furthermore, as compared to normal controls, the UALCAN database revealed that GTSE1 was substantially expressed in ccRCC samples at the protein level. (Figure 1E).
2.2 Overexpression of GTSE1 was shown to be associated with advanced clinicopathological characteristics.
We discovered no statistically significant difference in GTSE1 expression in patients with different ages (Figure 2A),but there were significant difference in different gender and high TNM, histological grade, or clinical stage of ccRCC (Figure 2B-G). GTSE1 overexpression was found to be significantly associated with pathologic stage (stage III vs stage I OR = 1.95, P<0.01; stage IV vs stage I OR = 4.896, P<0.001), histologic Grade (Grade 4 vs Grade 1 = 7.368, P 0.01); T classification (T3 vs T1 OR = 2.875 , P<0.001; T4 vs T1 OR=2.788, P = 0.011); N classification (N1 vs N0 OR= 15.126 P<0.001); and M classification (M1 vs M0 OR =2.436, p<0.001)(Table 1) .The high expression of GTSE1 was connected to histological grade (p<0.001), TNM stage (p<0.01), and pathologic stage (p<0.001), according to chi square test analysis (Table 2).
2.3 Survival outcome and Cox regression analysis
All patients were split into low and high subgroups based on the median expression value of GTSE1. The predictive value of GTSE1 in patients with ccRCC was evaluated using Kaplan-Meier survival analysis, and a ROC curve was created. The results revealed that patients in the GTSE1 high expression subgroup had a worse overall survival rate than those in the low expression subgroup (P<0.001). (Figure 3A-B). Because of the significant number of patients with missing N stage and M stage data in the database, we omitted N stage patients from the analysis and deleted patients with missing M stage data to minimize statistical bias.GTSE1 overexpression was strongly related with poor prognosis (HR = 2.027, p0.001), according to the findings of cox regression analysis. Other clinical characteristics, such as age, histological grade, clinical stage, T stage, and M stage, were also shown to be associated with a poor prognosis (all P < 0.001). GTSE1 expression(p=0.014), age (P =0.001), histological grade (P=0.013), pathologic stage (P< 0.05), and distant metastasis (P < 0.001)were all shown to be independent variables impacting OS values in multivariate Cox regression analysis (Table 3).
2.4 MicroRNA prediction
The role of noncoding RNA (ncRNA) in gene expression control is well understood. To see if any ncRNAs influence GTSE1, we first hypothesized that the upstream miRNA may bind to GTSE1 and then detected 14 miRNAs. miRNA is negatively linked with GTSE1 based on its action mechanism in the control of target gene expression. As a result, an expression correlation analysis was performed. As the table 4 shown that GTSE1 is strongly negatively linked with hsa-mir-23b-3p, hsa-mir-338-3p, and hsa-mir-532-3p, whereas the other four miRNAs have no statistical expression association (Table 4). Finally, the expression of hsa-mir-23b-3p, hsa-mir-338-3p, and hsa-mir-532-3p in ccRCC was determined, as well as their predictive values (Figure 4A-E). The same as the number.Only hsa-mir-23b-3p and hsa-mir-532-3p are considerably lowered in ccRCC, and only hsa-mir-23b-3p is up-regulated, suggesting that only hsa-mir-23b-3p is favorably connected with patient prognosis. All of these data point to hsa-mir-23b-3p as the most likely GTSE1 regulatory miRNA in ccRCC.
2.5 Prediction and analysis of upstream lncRNA of hsa-mir-23b-3p
Using a SateBase database, the upstream lncRNA of hsa-mir-23b-3p was predicted next. hsa-mir-23b-3p predicted a total of 53 lncRNA. lncRNA may boost mRNA expression by competing with shared miRNA, according to the competing endogenous RNA (ceRNA) theory. As a result, lncRNA and miRNA should have a negative connection, while lncRNA and mRNA should have a positive association. We projected a negative association between lnRNA and hsa-mir-23b-3p based on the preceding parameters(Table 5). PVT1 was discovered to be substantially expressed in ccRCC after further investigation(Figure 5A-B). In terms of expression, survival, and association analyses, PVT1 / has-mir-23b-3p / GTSE1 axis appears to be the most promising upstream lncrna in ccRCC.
2.6 GTSE1 was positively correlated with immune cell infiltration in ccRCC
We utilized the Timer website to investigate the function of GTSE1 in the immune system. In ccRCC, the infiltration level of CD8 + T, CD4 + T, neutrophil, and dendritic cells reduced when the copy number of GTSE1 was increased, as shown in Fig.6a. Further correlation analysis reveals crucial information for the research of GTSE1 function and mechanism. As a result, the relationship between GTSE1 expression and immune cell penetration was investigated. In ccRCC, expression of GTSE1 was substantially linked with all immune cells studied, including B cells, CD8 T cells, CD4 T cells, macrophages, neutrophils, and dendritic cells(Figure 6B-G).
2.7 Correlation between the expression of GTSE1 and immune cell biomarkers in ccRCC
We used the GEPIA database to examine the expression connection between GTSE1 and ccRCC immune cell biomarkers in order to further investigate the role of GTSE1 in tumor immunity. Table 4 shows the relationship between GTSE1 and B cell biomarkers (CD19 and CD79a), CD8 T cell biomarkers (CD8a and CD8b), CD4 T cell biomarkers (CD4), M1 macrophage biomarkers (NOS2, IRF5 and PTGS2), M2 macrophage biomarkers (CD163, VSIG4 and MS4A4A), neutrophil biomarkers (ITGAM and CCR7). The favorable connection between GTSE1 and immune cell penetration is somewhat supported by these data(Table 6).
2.8 Relationship between GTSE1 and ccRCC immune checkpoint
The immune checkpoints PDCD1/PD-L1/PD-L2 and CTLA-4 are curcial in tumor immune escape. The relationship between GTSE1 and PDCD1, PD-L1, PD-L2, or CTLA-4 was investigated in light of GTSE1's potential carcinogenic effect in ccRCC. In ccRCC, GTSE1 expression was strongly positively linked with PD1, PD-L2, and CTLA-4 after purity correction, but not with PDCD1. Furthermore, we checked the GTPIA database and discovered that in ccRCC, GTSE1 showed a strong positive connection with PDCD1, PD-L1, and CTLA-4, but no significant correlation with PD-L2(Figure 7A-H). Other immune cell biomarkers, in addition to neutrophil biomarkers, were found to be more or less favorably linked with GTSE1, indicating a link between GTSE1 and immune cell infiltration.