Abstract

   NEKs are proteins that are involved in various cell processes and play
   important roles in the formation and development of cancer. However,
   few studies have examined the role of NEKs in the development of
   non-small-cell lung carcinoma (NSCLC). To address this problem, the
   Oncomine, UALCAN, and the Human Protein Atlas databases were used to
   analyze differential NEK expression and its clinicopathological
   parameters, while the Kaplan–Meier, cBioPortal, GEPIA, and DAVID
   databases were used to analyze survival, gene mutations, similar genes,
   and biological enrichments. The rate of NEK family gene mutation was
   high (> 50%) in patients with NSCLC, in which NEK2/4/6/8/ was
   overexpressed and significantly correlated with tumor stage and nodal
   metastasis status. In addition, the high expression of NEK2/3mRNA was
   significantly associated with poor prognosis in patients with NSCLC,
   while high expression of NEK1/4/6/7/8/9/10/11mRNA was associated with
   good prognosis. In summary, these results suggest that NEK2/4/6/8 may
   be a potential prognostic biomarker for the survival of patients with
   NSCLC.

   Subject terms: Cancer, Lung cancer, Tumour biomarkers

Introduction

   Globally, lung cancer remains a major problem affecting human health,
   and its morbidity and mortality are continuously
   increasing^[36]1,[37]2. According to GLOBOCAN statistics, over 2.09
   million new cases of lung cancer were reported in 2018, accounting for
   11.6% of the total cancer cases, and 1.76 million lung cancer-related
   deaths occurred, accounting for 18.4% of the total cancer deaths^[38]3.
   Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer
   cases^[39]4, of which 25%–30% are squamous cell carcinomas^[40]5 and
   40%–45% are adenocarcinomas^[41]6. Overall, the 5-year survival rate of
   lung cancer is relatively low (approximately 19%)^[42]2. The main
   reason for this is that most patients are diagnosed at later stages and
   receive insufficient treatment^[43]7. Some previous studies have shown
   that some biomarkers can contribute to the rapid clinical diagnosis of
   NSCLC^[44]8. Therefore, there is an urgent need to develop a prognostic
   marker for NSCLC with potential clinical value in order to provide new
   ideas and methods for its early diagnosis and treatment.

   NEKs, also known as NIMA-related kinases, consist of 11 different
   family members that have N-terminal catalytic domains and encode
   different serine/threonine kinases^[45]9. Except for NEK11, they can be
   divided into four groups according to their specificity for
   serine/threonine phosphate receptors and their preference for
   acid/basic residues other than the 3-hydrophobic residue, namely,
   NEK1/3/4, NEK6/7/9, NEK5/8, and NEK2/10^[46]10. NEKs are proteins that
   are involved in various cell processes, such as cell cycle, mitosis,
   cilia formation, and DNA damage response; participate in cell
   differentiation and maintenance of cell homeostasis; and play important
   roles in the formation and development of cancer^[47]11. For example,
   NEK2/5 is associated with cell death resistance^[48]12,[49]13, of which
   NEK2 is significantly associated with lung cancer^[50]14 and NEK5
   expression is associated with breast cancer^[51]15. However, these
   studies mainly focused on a gene belonging to the NEK family, and the
   mechanism of all members of the family in the development of NSCLC has
   not been reported.

   In this study, we aimed to comprehensively analyze the expression,
   mutation, and clinical prognosis of NEK family members in patients with
   NSCLC in order to explore their potential clinical values.

Materials and methods

   Analysis of differential expression and pathological parameters. In
   order to analyze the expression of NEKs in NSCLC, we obtained the data
   from the Oncomine and UALCAN databases and visualized the expression
   profiles. The Oncomine gene expression microarray dataset
   ([52]www.oncomine.org) is a genome-wide expression dataset and a
   Web-based data-mining platform^[53]16 that can be used to detect the
   mRNA expression of NEK family members in 20 types of tumors. Using
   “NEKs” as the key word, the analysis type was “cancer and normal tissue
   analysis,” the setting parameter was a P < 0.01, the fold change
   was > 1.5, the gene rank was 10%, and the data type was mRNA. In
   addition, we analyzed the expression of NEK mRNA and its relationship
   with clinicopathological parameters such as tumor stage and nodal
   metastasis status in patients with LUAD and LUSC using the UALCAN
   database ([54]http://ualcan.path.uab.edu)^[55]17.

   Immunohistochemical analysis. The Human Protein Atlas (HPA,
   [56]https://www.proteinatlas.org/) is a database for the systematic
   study of human proteomes based on immunohistochemical analysis^[57]18.
   In this study, we obtained the expression patterns of NEKs proteins in
   human normal lung tissues and lung cancer tissues from this database
   and evaluated them as high, medium, low, and not detected according to
   the staining intensity.

   Overall survival analysis. The Kaplan–Meier Plotter database
   ([58]https://kmplot.com/analysis/) collects the survival data from the
   GEO and TCGA databases. It can study more than 54,000 genes and 21
   types of cancers and analyze the prognosis of malignant tumors online.
   In this study, we selected "auto select best cutoff" as the cutoff
   value and used this database to analyze the prognostic value of NEKs in
   patients with NSCLC.

   Mutation analysis. The cBioPortal database
   ([59]https://www.cbioportal.org/)^[60]19 can be used for interactive
   exploration of multiple cancer genomics datasets. Its data come from
   the TCGA, Oncomine, and other data platforms and can be used to study
   somatic mutations, DNA copy number mutations, DNA methylation, and
   other gene types. In this study, we used this database to analyze the
   genetic mutations of NEKs; the selected data set was “lung
   adenocarcinoma/lung squamous cell carcinoma (LUAD/LUSC) (TCGA, Firehose
   Legancy),” the name of the sample was “sample with mRNA data (RNA Seq
   V2,” z-score threshold =  ± 1.8)”, and the gene name “NEKs” was entered
   to conduct the search.

   Bioinformatic analysis of similar genes. GEPIA 2.0
   ([61]http://gepia.cancer-pku.cn/) was used to determine 50 adjacent
   genes that were significantly related to NEK mutations in NSCLC
   patients^[62]20, while STRING11.0 database ([63]https://string-db.org/)
   was used to analyze these similar genes, whose screening condition was
   with an interaction score of 0.9^[64]21. The MCODE plug-in in the
   Cytoscape software (version 3.8.1; [65]www.cytoscape.org) was used to
   filter out important modules using the following filter conditions:
   “Degree cutoff = 2,” “Node score cutoff = 0.2,” “K-core = 2,” and “Max
   depth = 100.” In addition, GO, KEGG, and RECTOME enrichment analyses of
   NEKs and similar genes were performed using the DAVID database
   ([66]https://david.ncifcrf.gov/summary.jsp)^[67]22.

Results

   Overexpression of NEK mRNA in NSCLC. We used the Oncomine database to
   measure the mRNA expression levels of NEKs in 20 cancer tissues.
   Results showed that there was overexpression of NEK2/4/6/8, low
   expression of NEK1/3/7, and no expression of NEK5/9/10/11 in lung
   cancer (Fig. [68]1). In addition, multiple data sets showed a
   significant increase in the expression of NEK2/4/6/8mRNA in lung cancer
   tissues (Table [69]1). We used the UALCAN database to detect the
   expression of NEK mRNA and found that there was a significant
   overexpression of mRNA in NEK2/4/6/8 in patients with LUAD and NEK2
   mRNA in patients with LUSC (P < 0.01) (Fig. [70]2).

Figure 1.

   [71]Figure 1
   [72]Open in a new tab

   Transcriptional expression of NEKs in 20 different types of cancer
   diseases.

Table 1.

   Significant changes of NEKs expression in transcription level between
   NSCLC and normal lung tissues.
        Gene Fold Change P value  t-test References