Association of the Timeless Gene with Prognosis and Clinical Characteristics of Human Lung Cancer

(1) Background: As the most common malignant tumor type worldwide, it is necessary to identify novel potential prognostic biomarkers to improve the poor prognosis of lung cancer. The Timeless gene, a circadian rhythm-related gene, is associated with several types of cancer. However, studies analyzing the clinical significance of the Timeless gene in patients with lung cancer are currently limited. (2) Methods: In the present study, the expression levels and prognostic potential of the Timeless gene and its co-expressed genes in different subtypes of lung cancer were explored using multiple bioinformatics approaches. The correlations between the Timeless gene and its co-expressed genes were validated using A549 and NCI-H226 cells by transfecting them with expression vectors and analyses using Western blot and reverse transcription-quantitative PCR. (3) Results: The Oncomine and GEPIA database analyses indicated that the expression of the Timeless gene was significantly higher in lung cancer as compared to that in the normal tissue. Using the UALCAN database, significant differences in Timeless gene expression were determined among different stages of lung cancer and between genders. A Kaplan–Meier plotter analysis indicated that high expression of the Timeless gene was associated with poor overall survival (OS) and progression-free survival (PFS) of patients with lung cancer. In the cBioPortal and GEPIA database analyses, extra spindle pole bodies like 1 (ESPL1) was the top correlated gene of Timeless in patients with lung cancer. Similar to the Timeless gene, high expression of the ESPL1 gene was also associated with poor OS and PFS. Of note, overexpression of the Timeless gene increased the expression level of ESPL1 at both the mRNA and protein levels. (4) Conclusion: The present study explored the clinical significance of the Timeless gene and its correlated gene ESPL1 in patients with lung cancer, thereby providing a potential therapeutic target for lung cancer.


Introduction
Lung cancer is the most common malignant tumor type worldwide. Lung cancer ranked third among the most prevalent cancers and first among the most common causes of cancer-related death in Europe, with 388,000 deaths in 2018 alone [1]. The five-year survival of patients with lung cancer has also been low in the past decades. Despite the considerable progress in the diagnosis and treatment of cancers, the prognosis of lung cancer has not been improved, yet. Its poor prognosis urges researchers to identify the underlying molecular mechanisms of lung cancer, as well as the potential prognostic biomarkers and novel therapeutic strategies.
Timeless is a highly conserved protein-coding gene, which is involved in cell survival after damage or stress. This gene is also involved in the increase in DNA polymerase epsilon activity, and maintenance of telomere length and epithelial cell morphogenesis. It also has a role in the circadian auto-regulatory loop by interacting with the Period genes (Per1, Per2 and Per3), and others to downregulate the activation of Per1 via the CLOCK/ARNTL pathway [2]. Recent studies using cell lines, animal models and human tissues demonstrated that the Timeless genes serve as oncogenes, having roles in the development of numerous cancer types. For instance, knockdown of the Timeless gene was able to markedly inhibit the cellular proliferation rate in breast and cervical cancer cell lines [3]. In human colon cancer cell lines, the expression level of the Timeless gene was negatively correlated with the microRNA-139-5p level [4]. However, numerous studies on the role of the Timeless gene in the diagnosis and prognosis of patients with lung cancer are relatively superficial [5,6]. In the present study, the relationship between extra spindle pole bodies like 1 (ESPL1) and Timeless and their expression in lung cancer was further studied. Timeless may be useful as a diagnostic and prognostic marker for lung cancer and targeting it may be of high therapeutic value for this disease.
The present study analyzed the expression and clinical significance of Timeless and its co-expressed genes in the different subtypes of lung cancer using multiple biological databases. Subsequently, Timeless and its co-expressed genes were bioinformatically analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways' analyses. The present study may benefit researchers in deeply understanding the role of Timeless and its co-expressed genes in lung cancer.

Oncomine Database Analysis
The Oncomine database (https://www.oncomine.org/resource/login.html (accessed on 1 January 2020)) is a web-based data-mining platform containing cancer microarray data. This database was used to investigate the mRNA expression levels of the Timeless gene in lung cancer and adjacent normal tissues. The data were statistically compared. The cut-off criterion was set as p < 0.05 and fold-change (FC) > 2.

GEPIA Database
GEPIA (http://gepia.cancer-pku.cn/ (accessed on 1 January 2020)) is a newly developed interactive web server, which analyzes the RNA expression profiles of 9736 tumor samples and 8587 normal samples, extracted from The Cancer Genome Atlas (TCGA) and the GTEx projects using a standard processing pipeline. In the present study, only the median expression data of the Timeless gene in the tumor and normal samples were extracted from the GEPIA database and analyzed using the bodymap.

UALCAN Database
The UALCAN database (http://ualcan.path.uab.edu/ (accessed on 1 January 2020)) is also an interactive web portal for in-depth analyses of gene expression data from TCGA. This database was used to analyze the correlations between Timeless gene expression and clinicopathological parameters of patients with lung cancer, such as the cancer stage, age, gender and smoking habits of the patients.

Human Protein Atlas (HPA) Database
The HPA (https://www.proteinatlas.org/ (accessed on 1 January 2020)) is a multifunctional platform, which aims to map all the human proteins in cells, tissues and organs by integrating various omics technologies, such as antibody-based imaging, mass spectrometry-based proteomics, transcriptomics and systems biology. This database was used to search for the immunohistochemical (IHC) staining images of the Timeless gene in normal and lung cancer tissues.

Kaplan-Meier (K-M) Plotter
The K-M plotter (http://www.kmplot.com/ (accessed on 1 January 2020)) is capable of assessing the effects of any gene or gene combination on the survival of patients with breast, ovarian, lung, gastric, colon, prostate and 14 other types of cancer using Diagnostics 2022, 12, 2681 3 of 15 >50,000 samples, which are measured using gene arrays, RNA-seq or next-generation sequencing technologies. In the present study, this tool was used to evaluate the prognostic potential of the Timeless gene in the different subtypes of lung cancer.

cBioPortal for Cancer Genomics
The cBioPortal (https://www.cbioportal.org/ (accessed on 1 January 2020)) for Cancer Genomics provides the visualization and analysis of large-scale cancer genomics datasets, which may be downloaded for manual analysis. In this study, the lung squamous cell carcinoma and lung adenocarcinoma datasets (TCGA, provisional) were selected to investigate the genetic alterations, correlations and network analysis of the Timeless gene in patients with lung cancer. The results provided the 25 most frequently altered neighbor genes, among which several genes were positively correlated with Timeless gene expression in lung squamous cell carcinoma and lung adenocarcinoma.

Database for Annotation, Visualization and Integrated Discovery (DAVID)
The DAVID v6.8 (https://david.ncifcrf.gov/ (accessed on 1 January 2020)) comprises a full knowledge base, updated to the sixth version of the original web-accessible programs. DAVID provides a comprehensive set of functional annotation tools for researchers to understand the biological annotations of large gene sets. GO and KEGG pathway enrichment analyses were performed using the 25 most frequently altered neighbor genes of Timeless in lung cancer.

Protein-Protein Interaction Analysis
The Search Tool for the Retrieval of Interacting Genes and proteins (STRING: https: //cn.string-db.org/ (accessed on 1 January 2020)) is a database of known and predicted protein-protein interactions, including direct (physical) and indirect (functional) interactions. The interactions are predicted using computational algorithms based on the knowledge transfer between organisms and interactions extracted from other (primary) databases. This database was used to construct the protein-protein interaction network among the Timeless gene and the 25 most frequently altered neighbor genes in both squamous cell lung carcinoma and lung adenocarcinoma.

A549 and NCI-H226 Cell Culture and Transfection
The human lung adenocarcinoma and squamous carcinoma cell lines, A549 and NCI-H226 cells, respectively, were obtained from the Type Culture Collection of the Chinese Academy of Sciences. The A549 cells were cultured in DMEM (HyClone; Cytiva, Tokyo, Japan) supplemented with 10% FBS (HyClone; Cytiva) and penicillin (100 U/mL)/streptomycin (100 U/mL) and incubated at 37 • C in a humidified atmosphere with 5% CO 2 . The NCI-H226 cells were cultured in 10% RPMI 1640 medium (HyClone; Cytiva). The human TIMELESS cDNA ORF-cloned expression vector (cat. HG18793-UT) and empty control pCMV3-untagged vectors were obtained from SinoBiological (Beijing, China). The A549 and NCI-H226 cells were transfected with the expression or empty control vectors for 48 h using Lipofectamine ® 3000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Reverse Transcription-Quantitative (RT-q)PCR
The mRNA expression levels of the ESPL1 gene in the two lung cancer cell lines (A549 and NCI-H226) were analyzed. For this purpose, the total cellular RNAs were extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The extracted RNA samples were reverse transcribed into cDNA using the iScript cDNA Synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following the manufacturer's instructions. Subsequently, the mRNA expression of the ESPL1 gene was measured using qPCR with the following primer pair: forward, 5 -GCCCTAAAACTTACAACAAA-3 and reverse, 5 -AGACTCAAGCAAGAACAGAA-3 ). The RT-qPCR was performed using the iTaq Fast SYBR Green Supermix (Bio-Rad Laboratories, Inc.) under the following cycle conditions: Diagnostics 2022, 12, 2681 4 of 15 50 • C for 2 min, 95 • C for 20 s, (95 • C for 1 s, 60 • C for 20 s) × 40 repeats. The relative mRNA expression levels of the ESPL1 gene were determined using the 2 −∆∆Cq method. All the experiments were performed independently in triplicate.

Statistical Analysis
All of the data were presented as the mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). The differences between the experimental and control groups were analyzed for statistical significance by Student's t-test. p < 0.05 was considered to indicate statistical significance. The statistical results and methods of public databases are derived from the statistical software built into these databases and were not deliberately processed.

Expression Level of the Timeless Gene in Human Lung Cancer
The expression levels of the Timeless gene were displayed using the Oncomine and GEPIA databases. The mRNA expression levels of the Timeless gene in various cancer types using pooled analyses are presented in Figure 1A. The results revealed that the Timeless gene was overexpressed in breast, brain, cervical, colorectal, head and neck and lung cancer tissues as compared to their matched normal tissues. The median expression of the Timeless gene in the tumor and normal tissue samples was analyzed using the bodymap from the GEPIA database ( Figure 1B). The Oncomine database also revealed that the expression levels of the Timeless gene were significantly higher in small cell lung carcinoma [7], squamous cell lung carcinoma [7][8][9], large cell carcinoma [8] and lung adenocarcinoma [8,10] (Figure 2). Furthermore, IHC staining images, acquired from the HPA database, also confirmed that the expression levels of the Timeless gene were upregulated in squamous cell lung carcinoma and lung adenocarcinoma as compared to the matched healthy lung tissues, which further validated the results from the Oncomine database ( Figure 3). In the HPA database, we found that Timeless was localized in the cytoplasm. Furthermore, the expression of the Timeless gene in healthy lung tissues was from a sample of a female patient aged 67 years (A; patient ID, 2208). The expression of the Timeless gene in lung adenocarcinoma was from a sample of a male patient aged 67 years (B; patient ID, 4886), and the expression of Timeless in lung squamous cell carcinoma was from a sample of a female patient aged 61 years (C; patient ID, 4900).
(B; patient ID, 4886), and the expression of Timeless in lung squamous cell carcinoma was from a sample of a female patient aged 61 years (C; patient ID, 4900).

Expression of Timeless Gene and Clinicopathological Parameters of Patients with Lung Cancer
The UALCAN database was used to analyze the associations between Timeless gene expression and clinicopathological parameters of patients with lung cancer, including lung squamous cell lung carcinoma and lung adenocarcinoma. The associations of Timeless gene expression with lung squamous cell carcinoma were analyzed. The results revealed higher expression levels of the Timeless gene in patients with cancer stages 1, 2 and 3 as compared to those in patients with cancer stage 4 and normal tissues (p < 0.05; Figure 4). The gender-specific gene expression analysis suggested that the expression of the Timeless gene was significantly upregulated in male and female patients with lung squamous cell lung carcinoma as compared to the normal group (p < 0.05). For lung adenocarcinoma, the analysis indicated that the expression of the Timeless gene was significantly upregulated in male patients as compared to female patients and the normal group (p < 0.05). However, there were no significant differences among different age groups or ethnicities.

Expression of Timeless Gene and Clinicopathological Parameters of Patients with Lung Cancer
The UALCAN database was used to analyze the associations between Timeless gene expression and clinicopathological parameters of patients with lung cancer, including lung squamous cell lung carcinoma and lung adenocarcinoma. The associations of Timeless gene expression with lung squamous cell carcinoma were analyzed. The results revealed higher expression levels of the Timeless gene in patients with cancer stages 1, 2 and 3 as compared to those in patients with cancer stage 4 and normal tissues (p < 0.05; Figure 4). The gender-specific gene expression analysis suggested that the expression of the Timeless gene was significantly upregulated in male and female patients with lung squamous cell lung carcinoma as compared to the normal group (p < 0.05). For lung adenocarcinoma, the analysis indicated that the expression of the Timeless gene was significantly upregulated in male patients as compared to female patients and the normal group (p < 0.05). However, there were no significant differences among different age groups or ethnicities.
The prognostic potential of the Timeless gene was analyzed using the K-M plotter ( Figure 5). Among the patients with lung squamous cell carcinoma and lung adenocarcinoma, a high expression of the Timeless gene was associated with poor overall survival (OS) and progression-free survival (PFS) ( Figure 5A-D); however, in patients with lung squamous cell carcinoma, there was no significance ( Figure 5E,F). For both male and female patients, the increased Timeless gene was also associated with unfavorable OS. The Timeless gene was of prognostic value for patients with lung cancer, irrespective of their smoking habits.  The prognostic potential of the Timeless gene was analyzed using the K-M plotter ( Figure 5). Among the patients with lung squamous cell carcinoma and lung adenocarcinoma, a high expression of the Timeless gene was associated with poor overall survival (OS) and progression-free survival (PFS) (Figure 5A-D); however, in patients with lung squamous cell carcinoma, there was no significance ( Figure 5E,F). For both male and female patients, the increased Timeless gene was also associated with unfavorable OS. The Timeless gene was of prognostic value for patients with lung cancer, irrespective of their smoking habits.

Genetic Mutations, Correlations and Networks Analysis of the Timeless Gene in Patients with Lung Cancer
The genetic mutations, correlations and network analyses of the Timeless gene in lung squamous cell carcinoma and lung adenocarcinoma (TCGA, provisional) were performed using the cBioPortal tool. The results indicated that the Timeless gene was mutated in 10 out of 230 (4%) patients with lung adenocarcinoma and 3 out of 178 (2%) patients with lung squamous cell carcinoma. The co-expression analysis of the Timeless gene in the patients with lung squamous cell lung carcinoma and lung adenocarcinoma was performed using the cBioPortal tool and the GEPIA database (Figures 6 and 7). Both for lung squamous cell lung carcinoma and lung adenocarcinoma, ESPL1 and Timeless were highly co-expressed with a strong correlation. ESPL1 was the top correlated gene, which was also confirmed using the GEPIA database.  In order to confirm the correlations between ESPL1 and the Timeless gene, A549 and  In order to confirm the correlations between ESPL1 and the Timeless gene, A549 and In order to confirm the correlations between ESPL1 and the Timeless gene, A549 and NCI-H226 cells were transfected with Timeless expression vector ( Figure 8A,B). Overexpression of the Timeless gene significantly increased the expression of ESPL1 at both the mRNA and protein levels. Furthermore, a protein-protein interaction network for the Timeless gene and the 25 most frequently altered neighbor genes in both lung squamous cell lung carcinoma and lung adenocarcinoma was constructed by using the STRING and DAVID databases (Figure 8C). As presented in Figure 9, a GO enrichment analysis indicated that the Timeless gene and its altered neighboring genes were mainly enriched in DNA replication, regulation of signal transduction by p53 class mediator and DNA damage checkpoint in the category biological process. In the category cellular component, these genes were mainly enriched in the DNA replication factor C complex and Ctf18 RFC-like complex in the nucleoplasm. Furthermore, these genes were mainly enriched in DNA clamp loader activity, single-stranded DNA-dependent ATPase activity and DNA binding in the category molecular function. The KEGG enrichment analysis for the Timeless gene and its frequently altered neighboring genes indicated that the mismatch repair, circadian rhythm and DNA replication may be involved in the oncogenic pathogenesis of lung cancer. Furthermore, a protein-protein interaction network for the Timeless gene and the 25 most frequently altered neighbor genes in both lung squamous cell lung carcinoma and lung adenocarcinoma was constructed by using the STRING and DAVID databases ( Figure 8C). As presented in Figure 9, a GO enrichment analysis indicated that the Timeless gene and its altered neighboring genes were mainly enriched in DNA replication, regulation of signal transduction by p53 class mediator and DNA damage checkpoint in the category biological process. In the category cellular component, these genes were mainly enriched in the DNA replication factor C complex and Ctf18 RFC-like complex in the nucleoplasm. Furthermore, these genes were mainly enriched in DNA clamp loader activity, single-stranded DNA-dependent ATPase activity and DNA binding in the category molecular function. The KEGG enrichment analysis for the Timeless gene and its frequently altered neighboring genes indicated that the mismatch repair, circadian rhythm and DNA replication may be involved in the oncogenic pathogenesis of lung cancer.
The prognostic potential of ESPL1 was similar to that of the Timeless gene in lung cancer (Figure 11). Among the patients with lung squamous cell carcinoma and lung adenocarcinoma, high expression of ESPL1 was associated with poor OS and PFS [p = 4.1 × 10 −12 , hazard ratio (HR) = 1.57 (95% CI, 1.38-1.78) and p = 2.1 × 10 −9 , HR = 1.78 (95% CI, 1.47-2.15), respectively]. High expression of ESPL1 mRNA was also significantly associated with poor OS only in patients with lung adenocarcinoma [p = 1.

Discussion
The present study comprehensively analyzed the mRNA expression levels o Timeless gene and its most significantly correlated gene ESPL1 in different subtyp human lung cancer using multiple databases. First, a critical role of Timeless in th

Discussion
The present study comprehensively analyzed the mRNA expression levels of the Timeless gene and its most significantly correlated gene ESPL1 in different subtypes of human lung cancer using multiple databases. First, a critical role of Timeless in the expression profile and prognosis of lung cancer was indicated. The associations of Timeless gene expression with clinicopathological parameters, including the cancer stage, gender, prognosis, race and age of patients with lung cancer, were also presented in this study. The GO and KEGG enrichment analyses identified the functional terms and pathways related to the Timeless gene and the frequently altered neighbor genes. These signaling pathways, including mismatch repair, circadian rhythm and DNA replication, have been demonstrated to have important roles in the oncogenic mechanisms of lung cancer. For instance, among the patients with non-small cell lung cancer, the genetic polymorphisms in the mismatch repair pathway may be potential clinical markers for the prediction of chemotherapeutic toxicity [13]. Furthermore, disruption of the circadian rhythm was also reported to be a potential risk factor for cancer development and poor prognosis, indicating the inhibitory effect of circadian rhythm homeostasis on the tumor [14].
The Timeless gene has an important role in the control of DNA replication, maintenance of replication fork stability, maintenance of genome stability throughout normal DNA replication and regulation of the circadian clock [15][16][17][18][19]. Given its potential role in the determination of period length and DNA damage-dependent phase advancing of the circadian clock, numerous studies have explored the correlations between the Timeless gene and tumorigenesis [20][21][22][23][24]. Chi et al. [21] demonstrated that the expression levels of the Timeless gene in both the breast cancer tissues and cell lines increased significantly. Knockdown of the Timeless gene was able to reduce the tumorigenicity of breast cancer cells in vivo, which may involve the well-known oncogene MYC [21]. These results were consistent with those of the previous studies on breast cancer [25][26][27]. This may provide a basis for using this gene as a therapeutic target for breast cancer. Although numerous studies indicated that the Timeless gene was overexpressed in cervical cancer, studies focusing on the underlying mechanism of this gene's role in cervical cancer are limited. Zhang et al. [24] suggested that the overexpression of the Timeless gene was associated with pelvic lymph node metastasis, demonstrating the role of the lympho-vascular space in human cervical cancer. This gene may have the potential of being used as an independent predictive biomarker for poor OS and PFS of patients with early-stage cervical cancer. However, its underlying molecular mechanisms are required to be explored further [24]. Similarly, studies focusing on the underlying mechanisms of the Timeless gene in lung cancer are also limited. A single-center study reported that the Timeless gene was overexpressed in surgically resected specimens among 88 consecutive patients. High protein levels of Timeless were associated with poor OS. Cytological experiments also confirmed that knockdown of this gene inhibited the growth of lung cancer cells and induced apoptosis in H157 and H460 cells [28]. Combining these results with those of the present study, it was demonstrated that the Timeless gene may be a valuable diagnostic and prognostic biomarker for lung cancer. Therefore, this gene should be focused on to explore its high therapeutic potential for lung cancer.
ESPL1 is a protein-coding gene and initiates the final separation of sister chromatids, which is a critical step for chromosomal inheritance [29]. Based on the GTEx database, ESPL1 is overexpressed in the esophagus-mucosa, vagina and testis. Current studies indicated that ESPL1 may also participate in the formation and progression of the tumor. For instance, as a cell cycle-associated gene, the overexpression of ESPL1 was significantly associated with a favorable prognosis. Furthermore, the expression level of ESPL1 was negatively associated with the pathologic stage/progression of gastric adenocarcinoma.
However, ESPL1/separase has been recognized as a putative oncogene of luminal B breast cancers [30]. In luminal breast cancers, the increased expression of ESPL1 was associated with poor prognosis of patients of the luminal B subtype. Animal experiments also verified that overexpression of ESPL1 in luminal tumors may be associated with the loss of tumor suppressor genes (P53 and Rb). In addition to lung cancer, studies also indicated that ESPL1 and five other genes were significantly upregulated in small cell lung carcinoma as compared to normal cells, lung squamous cell carcinoma, lung adenocarcinoma and large cell carcinoma. Furthermore, studies also suggested that overexpression of ESPL1 and the other five genes negatively affected OS and relapse-free survival [31]. Therefore, these genes may be used as powerful prognostic biomarkers and potential targets for patients with lung cancer. In order to confirm the association between the ESPL1 and Timeless genes, A549 and NCI-H226 cells were transfected with Timeless gene expression vectors. Overexpression of the Timeless gene significantly increased the expression level of ESPL1 at both the mRNA and protein levels. A549 and NCI-H226 cells represented lung squamous cell carcinoma and lung adenocarcinoma, respectively. The cell culture and transfection experiments reflected the association between the ESPL1 and Timeless genes, which preliminarily confirmed the significant role of these genes in the development of tumors. However, the regulatory regions and downstream regulatory elements are still required to be further studied. Altogether, these results suggested that the Timeless gene and its co-expressed ESPL1 gene may have great prognostic and treatment potential in lung cancer.
However, there are certain limitations to this study. First, the present study only explored the expression levels and prognostic potential of the Timeless and ESPL1 genes in lung cancer using several public databases. However, this study did not validate these findings using PCR or IHC. Furthermore, although the GO and KEGG enrichment analyses were able to screen out certain related pathways and biological processes, limited studies have explored the underlying mechanisms of the effects of Timeless on the prognosis of patients with cancer. Considering the limited funds available, the expressions of Timeless and ESPL1 were only determined in lung cancer cell lines. In future experiments, the effect of Timeless on the invasion and migration in lung cancer cells and normal cells from patients may be investigated to further confirm the clinical significance of Timeless in lung cancer.
In conclusion, the present study comprehensively analyzed the mRNA expression levels of the Timeless gene and its most significantly correlated gene ESPL1 in different types of human lung cancer and predicted the underlying mechanisms of these genes in lung cancer, thereby providing a better understanding of lung cancer pathogenesis. These results suggested that high expression of the Timeless and ESPL1 genes in lung cancer tissues may have an important role in the development of lung cancer. Therefore, these genes may serve as potential prognostic biomarkers for the improvement of survival of patients with lung cancer and prognostic accuracy.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets analyzed during the current study are available from the corresponding author upon reasonable request.