Abnormally high expression of TIMELESS in glioma.
First, we analyzed the expression of TIMELESS in a variety of tumors and corresponding normal tissues through the GEPIA tool, and found that TIMELESS was widely high expression in BLCA, BRCA, CESC, COAD, DLBC, GBM, LGG, LUSC, OV, READ, SKCM, STAD, TGCT, THYM, UCEC, UCS, but was the low expression in LAML(Fig. 1A).
To further study the mechanism of TIMELESS in the tumor, we chose glioma as the research object. Through GSE4290 (77 glioma tissue samples and 23 normal brain tissue samples), GSE50161 (34 glioma tissue samples and 13 normal brain tissue samples), and GSE11652 (34 glioma tissue samples and 8 normal brain tissue samples) database analysis, we again verified that TIMELESS was indeed highly expressed in glioma (Fig. 1B-D, p < 0.001). Through the mutual verification of multiple data sets, we can effectively improve the credibility of our data results. This study will lay a foundation for further study of the mechanism of TIMELESS in glioma.
High expression of TIMELESS was an unfavorable factor for the prognosis of glioma patients.
In order to evaluate the relationship between the high expression of TIMELESS and the prognosis of glioma patients, the original data from the CGGA RNA-seq, CGGA microarray, and TCGA RNA-seq databases were divided into a high expression group and a low expression group according to the expression of TIMELESS. Firstly, Compared with the low expression group of TIMELESS in 376 glioma patients through the Kaplan-Meier method, the high expression of TIMELESS in 372 glioma patients showed that the survival time of patients in the high expression group was significantly lower than that in the low expression group based on the CGGA RNA-seq (Fig. 2A). Secondly, Compared with the low expression group in 134 glioma patients through the Kaplan-Meier method, the high expression of TIMELESS in 134 glioma patients showed that TIMELESS can also reduce the survival time of glioma patients based on the CGGA microarray (Fig. 2B). Finally, in order to test the effect of TIMELESS expression in different ethnic groups on the prognosis of patients, we further verified the impact of high expression of TIMELESS on the prognosis of patients in TCGA RNA-seq, because the dataset mainly contains American race, while CGGA database contains Chinese race. It was found that the increased expression level of TIMELESS could significantly reduce the overall survival of glioma patients including 653 glioma patients (Fig. 2C). The results were surprisingly consistent with the CGGA database. However, whether high expression of TIMELESS was an independent factor for poor prognosis requires further study.
High expression of TIMELESS was an independent factor for poor prognosis of glioma patients
We used univariate and multivariate analysis to analyze the relationship between clinical sample information and the survival status of glioma patients. From the analysis of the common results in the above three databases, we found that TIMELESS expression and glioma grade is closely related to the prognosis of glioma patients (Fig. 3A-3F). The detailed results are as follows: in univariate analysis, TIMELESS expression and patient survival time have significant statistical significance in CGGA RNA-seq (HR: 1.733, 95% CI: 1.587–1.892, p < 0.001, Fig. 3A), CGGA microarray (HR: 1.902; 95% CI: 1.636–2.212, p < 0.001, Fig. 3C) and TCGA RNA-seq (HR: 1.087, 95% CI: 1.064–1.109, p < 0.001, Fig. 3E). In multivariate analysis, it also has statistical significance in CGGA RNA-seq (HR: 1.252, 95% CI: 1.137–1.379, p < 0.001, Fig. 3B), CGGA microarray (HR: 1.483, 95% CI: 1.234–1.783, p < 0.001, Fig. 3D) and TCGA RNA-seq (HR: 1.034, 95% CI: 1.004–1.065, p < 0.05, Fig. 3F). At the same time, in univariate analysis, glioma grade and patient survival time have significant statistical significance in CGGA RNA-seq (HR: 2.883, 95% CI: 2.526–3.291, p < 0.001, Fig. 3A), CGGA microarray (HR: 2.567; 95% CI: 2.125–3.100, p < 0.001, Fig. 3C) and TCGA RNA-seq (HR: 4.634, 95% CI:3.727–5.760, p < 0.001, Fig. 3E). In multivariate analysis, it also has statistical significance in CGGA RNA-seq (HR: 2.506, 95% CI: 1.825–3.441, p < 0.001, Fig. 3B), CGGA microarray (HR: 2.402, 95% CI: 1.389–4.155, p < 0.005, Fig. 3D) and TCGA RNA-seq (HR: 3.044, 95% CI: 2.401–3.860, p < 0.001, Fig. 3F). But, due to incomplete clinical data in the TCGA RNA-seq database and the HR value of the age crosses the invalid line in the CGGA microarray multivariate analysis, whether PRS type and age can be used as an independent factor for glioma patients requires further analysis. In conclusion, TIMELESS High expression is an independent factor for poor prognosis of glioma patients.
TIMELESS high expression has diagnostic value for patient prognosis
To explore whether the high expression of TIMELESS has diagnostic value for the prognosis of glioma patients, we analyzed the above three sets of data and by cox regression. the high expression of TIMELESS as an independent factor for glioma patients with a poor prognosis has clinical diagnostic value in 1 year, 3 years, and 5 years from CGGA RNA-seq (Fig. 4A), CGGA microarray (Fig. 4B), and TCGA RNA-seq (Fig. 4C). The results suggest that TIMELESS can be used as a biomarker for the diagnosis and individualized treatment of glioma.
The relationship between TIMELESS and clinical features of poor prognosis in patients with glioma.
The results of Wilcox or Kruskal test correlation analysis showed that the abnormally high expression of TIMELESS was positively correlated with WHO grade, PRS type, chemo status, and histology (Fig. 5A, C, D, G, p < 0.001), but IDH1 mutation and 1p19q codeletion states were negatively correlated (Fig. 5E, F, p < 0.001) in the data of CGGA RNA-sEq. Meanwhile, it was positively correlated with WHO grade, histology, and negatively correlated with IDH1 mutation (Fig. 5A, E, G, p < 0.001) in CGGA microarray. Finally, it also was positively correlated with WHO grade, age in the data of TCGA RNA-seq (Fig. 5A, B, p < 0.001). By analyzing the above three databases, we found that the low expression of TIMELESS is closely related to low tumor grade, 1p19q joint deletion, and IDH1 mutation. As our clinical knowledge knows, 1p19q joint deletion, IDH1 mutations often occur in low-grade glioma, and patients often have a relatively good prognosis. Therefore, it further confirms indirectly supports that TIMELESS was a carcinogenic gene in glioma. However, the mechanism by which TIMELESS participates in the malignant behavior of glioma requires my next to reveal.
Cell signaling pathways of TIMELESS in glioma
We divided the tissue samples into the high expression group and low expression group based on the expression level of TIMELESS. Then, GSEA analysis was used to reveal the activation of cancer-related cell signaling pathways caused by high expression of TIMELESS. Potential cellular molecular pathways were selected out by the consistent results of three databases together enriched (Table 1). The TIMELESS high expression may participate in tumor development by activating cell cycle, mismatch repair, and DNA replication cell signaling pathways (Fig. 6A-C).
Table 1
The gene set enriches the high TIMELESS in three databases.
GENE SET NAME | CGGA RNA-seq | CGGA microarray | TCGA RNA-seq |
NOM p-value | FDR q-value | NOM p-value | FDR q-value | NOM p-value | FDR q-value |
CELL CYCLE | 0.000 | 0.061 | 0.000 | 0.000 | 0.000 | 7.52E-04 |
DNA REPLICATION | 0.000 | 0.108 | 0.000 | 0.018 | 0.000 | 0.005 |
MISMATCH REPAIR | 0.002 | 0.113 | 0.008 | 0.043 | 0.000 | 0.005 |
BASE EXCISION | 0.002 | 0.203 | 0.039 | 0.125 | 0.000 | 0.005 |
HOMOLOGOUS RECOMBINATION | 0.006 | 0.180 | 0.000 | 4.00E-04 | 0.000 | 0.005 |
Gene sets with NOM P-value < 0.05 and FDR q-value < 0.25 were considered as significantly enriched, NOM: nominal; FDR: false discovery rate. |
Abnormally high expression of TIMELESS protein levels in glioma
We have verified the abnormally high expression of TIMELESS in glioma from the level of mRNA. However, the level of TIMELESS protein is unclear. So, we downloaded the immunohistochemical film of TIMELESS protein expression in glioma tissue and normal brain tissue from The Human Protein Atlas website. Then divided into three groups according to tumor grade, including the normal group, low-grade group, high-grade group. We found that the TIMELESS protein expression level was markedly higher in both the low-level group and the high-level group than the normal group (Fig. 7).
Drugs of potential inhibit TIMELESS
In order to find drugs that potentially inhibit expression of TIMELESS, we first performed a correlation analysis between TIMELESS and other differential genes. Select 10 positively related genes and 10 negatively related genes and draw a circle diagram (Fig. 8). Next, we uploaded these 20 related genes to the CMap website and screened out 4 small molecule compounds that potentially inhibit TIMELESS, such as 8-azaguanine, gw8510, 6-Thioguanosine, and ursodeoxycholic acid (Fig. 9, Table 2). Some of these drugs have been found to have clear anti-tumor effects in other studies. This reflects the reliability of our predictions.
Table 2
Small molecule compounds predicted by CMap
NO. | CMap name | enrichment | p |
1 | GW-8510 | -0.929 | 0.00002 |
2 | roxithromycin | -0.904 | 0.00016 |
3 | ursodeoxycholic acid | -0.885 | 0.003 |
4 | thioguanosine | -0.882 | 0.00048 |
5 | 8-azaguanine | -0.786 | 0.00426 |
Enrichment<-0.7, p < 0.05. connectivity map: CMap |