Analysis of the effect of NEKs on the prognosis of patients with non-small-cell lung carcinoma based on bioinformatics

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.


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. 1). In addition, multiple data sets showed a significant increase in the expression of NEK2/4/6/8mRNA in lung cancer tissues (Table 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. 2).
Overexpression of NEK proteins in NSCLC. We used the HPA database to explore the protein expression levels of NEKs in NSCLC. We found that NEK2 protein was not expressed in normal tissues and NSCLC, and the protein expression levels of other NEKs in NSCLC showed higher expression levels than those in normal tissues. The NEK3/5/7/10/11 proteins showed no expression in normal tissues, but low and moderate expression in NSCLC. The NEK1/4/6/8/9 proteins showed low and medium expression in normal tissues, but high and medium expression in NSCLC (Fig. 3).
Analysis of the relationship between NEKs and clinicopathological parameters. We used the UALCAN database to analyze the relationships between NEK mRNA expression levels and the bedside pathological parameters of NSCLC patients, including tumor stage and nodal metastasis status. With regard to tumor stage, the expression of NEK mRNA was significantly correlated with the tumor stage of patients. Results showed that in LUAD patients, the highest expression levels of NEK1/2/3/4/7/9/10/11 mRNA were observed in those with stage 4, while the highest expression levels of NEK5/6/8 mRNA were observed in those with stages 3, 2, and 1, respectively (Fig. 4A). Meanwhile, patients with LUSC had the highest second-phase mRNA expression levels of NEK1/2/8/9/10 and the highest fourth-phase mRNA expression levels of NEK3/4/5/6/7/11 (Fig. 4B). In terms of nodal metastasis status, NEK2/4/6/8 mRNA was overexpressed in LUAD patients, and their highest expression levels were in N3 (Fig. 5A). In patients with LUSC, only the mRNA of NEK2 was overexpressed (Fig. 5B).
Prognostic values of NEK mRNA expression in NSCLC patients. We used the Kaplan-Meier plotter database to analyze the effect of NEK mRNA expression on the prognosis of patients with NSCLC. Results showed that the expression of NEK mRNA was significantly correlated with the prognosis of patients with NSCLC, but NEK5 could not find the related prognostic information. Among them, the high mRNA expression of NEK family members is related to the long-term prognosis of NSCLC patients (hazard ratio [ www.nature.com/scientificreports/ NEKs gene mutations. We used the cBioPortal database to analyze the gene mutations of NEKs. Among the 515 LUAD patients, 288 patients had gene mutations, and the mutation rate was 56%; in 501 LUSC patients, 267 had gene mutations, and the mutation rate was 53%. Among LUAD patients, the mutation rates of NEK/2/7/9 were the highest, at 15%, 15%, and 14%, respectively. Among the patients with LUSC sequencing, the NEK2/9/11 gene mutation rates were the highest, at 14%, 11%, and 15%, respectively (Fig. 7).
Functional enrichment analysis of NEKs and similar genes. We used GEPIA 2.0 to search 50 adjacent genes that were significantly associated with NEK mutations in NSCLC patients and STRING11.0 database and Cytoscape to screen and visualize important module genes (Fig. 8A). Among them, CDK1, PLK1, CCNB1, and CCNB2 were significantly correlated with NEK mutations.
We used the DAVID database to analyze the NEKs and similar genes. Results of GO enrichment showed that molecular functions (MF), such as GO:0,003,777 (microtubule motor activity), biological processes (BP) such as GO:0,007,062 (sister chromatid cohesion), and cellular components(CC) such as GO:0,000,776 (kinetochore) were significantly regulated by NEK mutations in NSCLC (Fig. 8B). In addition, the pathway enrichment analysis  www.nature.com/scientificreports/ showed that hsa04110 (cell cycle) and hsa04914 (progesterone-mediated oocyte maturation) were significantly related to the functions of NEKs in NSCLC (Fig. 8C).

Discussion
The occurrence and development of lung cancer is a multi-stage process involving multiple genes and factors 31 .
In addition to cancer genetics, abnormal epigenetic regulation at all stages plays an important role in its development 32 . It can be used as a reliable tool for early diagnosis and prognosis monitoring and as an effective therapeutic target for patients with lung cancer 33 . Although the roles of some members of the NEK family in Figure 2. mRNA expression of distinct NEKs family members in NSCLC tissues and and normal lung tissues. *P < 0.05, **P < 0.01, ***P < 0.001. A From a to k represent the mRNA expression of NEK1 to NEK11 in LUAD tissues and normal lung tissues; B From a to k represent the mRNA expression of NEK1 to NEK11 in LUSC tissues and normal lung tissues. www.nature.com/scientificreports/ lung cancer have been confirmed, investigating individual gene expression levels is not enough to evaluate the mechanism of lung cancer; therefore, the different roles of NEK family members in lung cancer still need to be clarified. In this study, we analyzed the differential expression, pathological parameters, mutations, and prognostic values of different NEK family members in NSCLC.
Results of this study showed that the NEK2/4/6/8 mRNA was overexpressed in LUAD patients, both in tumor stage and nodal metastasis status, and the highest expression level was mainly concentrated in phase 3/4; NEK2 mRNA was also overexpressed in LUSC patients, and the highest expression level was in phase 4; Therefore, we believe that the mRNA expression level of the above genes increases with the increase of tumor stage and nodal metastasis status. According to the clinical guidelines, the later the tumor stage and nodal metastasis status, the worse the prognosis. Therefore, it is speculated that NEK2/4/6/8 may become potential prognostic biomarkers for the survival of NSCLC patients. In addition, the expression levels of NEK1/3/4/5/6/7/8/9/10/11 proteins in NSCLC were higher than those in the normal group, which may be related to carcinogenesis, but NEK2 protein showed no expression in both normal group and NSCLC group, which may be due to the low expression levels of immune cells infiltrated in NSCLC. Furthermore, the high expression of NEK2/3 in NSCLC patients was significantly correlated with shorter overall survival time (OS), while the high expression of NEK1/4/6/7/8/9/10/11 mRNA in NSCLC patients was significantly correlated with good OS, which may be due to the fact that genes with high expression levels are more sensitive to treatment; hence, the prognosis is better. In addition, we observed high mutation rates of 56% and 53% in patients with LUAD and LUSC, respectively. NEK2/9 showed higher mutation in LUAD and LUSC patients, and mutation is a reason for the change of mRNA expression. Therefore, we speculated that NEK2/9 may interfere with mRNA expression by affecting DNA expression level and indirectly affect the prognosis of patients. Finally, CDK1, PLK1, CCNB1, and CCNB2 were significantly associated with NEK mutations. The GO enrichment analysis showed microtubule motor activity, ATP binding, and protein serine/threonine kinase activity in MF; sister chromatid cohesion, microtubule-based movement, and antigen processing and presentation in BP; and kinetochore, microtubule, and condensed chromosome kinetochore in CC. KEGG and RECTOME enrichment analysis showed that the main pathways were cell cycle-and progesterone-mediated oocyte maturation, which were significantly related to the functions of NEKs in NSCLC.
NEK2 is involved in mitosis and in the regulation of centrosome replication and spindle formation 34 . When the regulation is abnormal, chromosomal instability and aneuploidy occur, which are landmark changes in many tumors 35,36 , indicating that it plays an important role in the occurrence and development of tumors. At present, NEK2 is overexpressed in many cancers and is related to the poor prognosis of pancreatic ductal cancer 37 , prostate cancer 38 , and colon cancer 39 . In NSCLC patients with EGFR mutations, EGFR mutations can activate the ERK signal transduction pathway and then induce the high expression of NEK2, which can promote the rapid development of the cell cycle and the rapid proliferation of NSCLC cells, resulting in poor prognosis 40 . qRT-PCR experiments also showed that the expression level of NEK2 in LUAD tissues increased with the increase in TNM stage, and the high expression of NEK2 was significantly correlated with poor prognosis 41 . In this study, NEK2 was overexpressed in NSCLC, and it was significantly correlated with tumor stage, nodal metastasis status, and poor prognosis; this finding is consistent with the conclusions of the abovementioned study, indicating that NEK2 is more likely to be used as a prognostic marker of NSCLC, but further research is needed.
In the process of cancer development, NEK4 can repair defects by inhibiting DNA, which makes cancer cells sensitive to apoptosis 42 43 . In addition, the lack of NEK4 can reduce the expression of survivin 44 ; survivin is an anti-apoptotic protein highly expressed in most cancers, which can increase the tumor recurrence rate and reduce the survival rate of patients 38,45 . NEK6 can regulate many cell processes, such as cell cycle, apoptosis, and senescence 46 , and accelerate the entry of tumor cells in the S phase 47 . The expression level and activity of NEK6 in mitosis of many types of malignant tumors can be increased 48,49 , which can inhibit the premature senescence of cancer cells induced by p53 and promote the occurrence and development of tumors 47 . Previous studies have shown that NEK6 is significantly upregulated in breast cancer and may play a role in the proliferation of breast cancer cells. In addition, the expression of NEK6 was positively correlated with histological grade, tumor size, and TNM stage of breast cancer; therefore, we believe that NEK6 may be an important index for predicting the prognosis of patients with breast cancer 46 . Some studies have also shown that NEK6 is overexpressed in advanced gastric cancer and in 70% of liver cancer patients 50,51 . Animal experiments have also shown that low expression levels of NEK6 can lead to tumor cell reduction 52 . www.nature.com/scientificreports/ NEK8 plays a role in the cell cycle G2-M phase 53 and the DNA damage response signaling pathway 53,54 . It can play a potential role in tumorigenesis and DNA damage response through the MYC proto-oncogene 55 and serine/threonine kinase signaling pathway 54 . NEK8 may be the target gene of hypoxia-inducible factor (HIF), whereas von Hippel-Lindau tumor suppressor protein may downregulate the expression of NEK8 through the HIF 56 . In addition, some studies have found that NEK8 is overexpressed in breast cancer 57 .
In this study, NEK4/6/8 was highly expressed in LUAD, which was closely related to tumor stage and nodal metastasis status. However, their high expression levels were associated with good prognosis, probably because the high expression level made these genes more sensitive to treatment; hence, the prognosis was better. NEK1/10/11 is related to DNA damage response 34 . Among them, the expression level and activity of NEK1 are increased during mitosis 58,59 . When it is mutated or silenced, spindle defects, abnormal chromosome segregation, mitotic block, and apoptosis occur; therefore, NEK1 plays an important role in the development of cancer 34,[58][59][60] . NEK1 can participate in the regulation of DNA damage repair in HeLa cervical cancer cells 61 , and its overexpression can significantly reduce disease-free survival 62 ; it can also be overexpressed in human glioma tissues, which is related to tumor grade and nodal metastasis status 63 . A decrease in NEK10 expression can lead to increased cell proliferation and DNA replication 64 , which is related to poor prognosis and higher tumor grade of breast cancer 65 . As part of the physiological barrier induced by DNA damage, NEK11 was overexpressed in precancerous lesions of 35% of colorectal adenomas and colon cancer, and its high expression level is associated www.nature.com/scientificreports/ with low tumor grade and weak invasive ability 66,67 . NEK11 is a potential tumor suppressor gene 68 and is significantly downregulated in ovarian cancer mediated by methylation or mutation 69,70 . In this study, however, the low expression of NEK1 in NSCLC was significantly correlated with poor prognosis, which was inconsistent with the above results, which may be due to the decrease in the overall average expression level of immune cells with low expression levels in NSCLC. The results of NEK10/11 are consistent with those of the abovementioned study; the higher the expression levels, the better the prognosis. Some studies have found that NEK3 can regulate cell migration, proliferation, viability, and neuronal development [69][70][71][72] and may play an important role in the occurrence and development of cancer 73 . The expression level of NEK3 is related to the invasiveness, TNM stage, and tumor size of thyroid carcinoma 14 . In addition, NEK3 is overexpressed in gastric cancer, which is closely related to pT stage, TNM stage, and nodal metastasis status, and can significantly reduce OS and disease-free survival in patients with gastric cancer 74 . NEK3 is also highly expressed in invasive breast cancer, which can promote the migration and invasion of breast cancer, and inhibition of its activity can lead to loss of invasive phenotype 75 . However, in this study, the low expression of NEK3 in NSCLC may be due to the loss of expression during the transformation of lung malignant tumors 16 , and its high expression level is significantly correlated with shorter OS. Some studies have suggested that NEK5 is also related to the development of cancer and can interact with caspase-3 76 . Caspase-3 is a protease involved in cell apoptosis and differentiation; therefore, NEK5 is also involved in cell death 13,76 . The upregulation of NEK5 is significantly related to the progression and poor prognosis of breast cancer. When NEK5 is silenced, it can significantly prevent the proliferation of breast cancer cells in vivo and in vitro, thus inhibiting their migration and invasion 15 . In this study, we found that the expression level of NEK5 in NSCLC is relatively low; moreover, no study has investigated its prognostic value. Due to the lack of research on NEK5, its importance in the occurrence and development of cancer cannot be confirmed. NEK9/7/6 can be activated during mitosis and jointly regulate spindle formation 77 , in which activated NEK9/7 can change the microtubule dependence of NSCLC cell morphology and promote its migration 78 . NEK9 is www.nature.com/scientificreports/ thought to play a role upstream of NEK7/6 79 , and its main function is to activate NEK7/6 through various mechanisms such as activated cyclic phosphorylation, autophosphorylation induced by dimerization, and catalytic site recombination induced by allosteric binding [79][80][81][82] . For example, NEK9 can directly interact with NEK7 through a short sequence (residues 810-828) located in its C-terminal region between the RCC1-like domain (residues 347-726) 78 and the RCC1-like domain C-terminal coiled helix motif (residues 891--939) 83 and may interact with NEK6 81 . The carcinogenic driver EML4-ALK 84 is detected in 5% of NSCLC patients, and the high expression of NEK9 can lead to poor progression-free survival 78 . NEK7 can be highly expressed in gallbladder carcinoma, which is closely related to tumor differentiation, Nevin stage, and metastasis, and can significantly shorten the OS 85 . At present, a few studies have investigated the expression levels, prognostic values, and interactions of NEK9/7 in tumor cells; in this study, after analyzing the expression levels of NEK9/7 in NSCLC and normal tissues, it was found that NEK9/7 is highly expressed in normal tissues and is closely related to a good prognosis. In summary, our results show that the mutation rates of NEK family genes in NSCLC are relatively high. In LUAD patients, the mutation rate was 56%, in which NEK2/4/6/8 genes were overexpressed. In LUSC patients, the mutation rate was 53%, in which NEK2 was overexpressed; all of them were significantly related to 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 the high expression of NEK1/4/6/7/8/9/10/11 mRNA was associated with good prognosis. These results suggest that NEK2/4/6/8 may be a prognostic biomarker for the survival of patients with NSCLC. However, since this study only evaluated the relationship between NEK mRNA expression level and survival rate and did not consider the limitations of other confounding factors, the results of survival analysis are still uncertain, and further studies are needed to verify these results. www.nature.com/scientificreports/

Data availability
All data generated or analyzed during this study are included in this published article. www.nature.com/scientificreports/