NEK2 could act as a circadian time-dependent tumor suppressor gene and biomarker in esophageal squamous cell carcinoma

Abstract

Background As one of the most prevalent cancers worldwide, esophageal squamous cell carcinoma (ESCC) is characterized by rapid progression and poor prognosis, thus, it is essential to develop novel biomarkers to evaluate the diagnosis and prognosis of ESCC patients. NIMA-related kinase 2 (NEK2) is an oncogenic kinase that has been found to be upregulated in a variety of cancers, and is considered a potential therapeutic target. In addition, circadian rhythms have been shown to be closely related to the development of cancer. However, the relationship between NEK2, circadian rhythm and ESCC progression remain unknown. Methods The Cancer Genome Atlas (TCGA), and Gene Expression Omnibus (GEO) databases (GSE23400 and GSE53625) were employed to collected the gene expression in ESCC patients, gene expression data, and clinical information. Several bioinformatics methods, including the Gene Set Enrichment Analysis (GSEA) and survival analysis were utilized to explore the correlation between the expression patterns of NEK2 genes and the development of ESCC. RNA sequencing was performed to investigate the expression of circadian rhythm. The effects of INH6 on the growth and proliferation of ESCC cell lines were tested in vitro by the Cell Counting Kit 8 (CCK8), 5-ethynyl-2ʹ-deoxyuridine (EdU), and colony formation. Additionally, western blotting was used to examined the expression of NEK2, cell cycle and apoptosis related protein, such as P21and cleaved PARP. reverse transcription-quantitative PCR (RT-qPCR) was employed to confirm that core circadian gene Bmal1, and Rev-Erba expressed. The cell apoptosis was detected by flow cytometry. Results Here, we first found that NEK2 gene was identified as differentially expressed genes in both the three datasets. Additionally, NEK2 is overexpressed in ESCC tissues and inhibition of NEK2 impairs ESCC cell growth and proliferation. The analysis of the KEGG and GSEA pathways revealed that NEK2 is closely related to known and recognized pathways such as the cell cycle, but an interesting new pathway related to NEK2 was also identified: circadian rhythm. RNA sequencing identified that NEK2 exhibits circadian rhythmic expression, and further in vitro studies confirmed that NEK2 has circadian oscillation in synchronized ESCC cells. More importantly, we observed that INH6 efficacy correlated positively with NEK2 expression levels in ESCC, suggesting that it is well worthwhile to develop and optimize the in vivo efficacy evaluation of NEK2 inhibitors based on chronological administration. Conclusions According to this study, NEK2 is highly expressed in human EC, and NEK2 has circadian oscillation in synchronized ESCC cells, which could be used to increase INH6 response to esophageal cancer therapy. In a word, NEK2 acts as a time-dependent tumor suppressor gene and biomarker in ESCC.