| Summary: | (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 (<i>ESPL1</i>) was the top correlated gene of Timeless in patients with lung cancer. Similar to the Timeless gene, high expression of the <i>ESPL1</i> gene was also associated with poor OS and PFS. Of note, overexpression of the Timeless gene increased the expression level of <i>ESPL1</i> at both the mRNA and protein levels. (4) Conclusion: The present study explored the clinical significance of the Timeless gene and its correlated gene <i>ESPL1</i> in patients with lung cancer, thereby providing a potential therapeutic target for lung cancer.
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