3. 1 Pan-cancer analysis of NEK2 expression
We extracted mRNA levels of NEK2 from 33 cancer types in the TCGA database and plotted box plots of NEK2 expression in cancerous tissues and normal tissues adjacent to the cancer. NEK2 mRNA expression was significantly upregulated in most cancers, including BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, KICH, KIRC, KIRP, LIHC, LUAD, LUSC, PAAD, PCPG, PRAD, READ, SARC, STAD, THCA, and UCEC, with no significant difference in SKCM and THYM (Fig. 1A). Since the transcript levels of the corresponding paracancerous normal tissues in ACC, DLBC, LAML, LGG, MESO, OV, TGCT, UCS, and UVM were not available, we integrated the normal tissue data into the GTEx database data. As shown in Fig. 1B, NEK2 mRNA levels were also higher in ACC, DLBC, LGG, OV, SKCM, THYM and UCS, and NEK2 mRNA expression levels were lower in TGCT. In addition, Fig. 1C shows the relative expression levels of NEK2 in various cancer cell lines in the CCLE database. As can be seen from the results, NEK2 is usually expressed at higher levels in tumor cell lines from 33 tissues. These results indicate that NEK2 expression is up-regulated in many types of cancers, suggesting that NEK2 may play a key role in cancer diagnosis and treatment.
In addition, we investigated the expression level of NEK2 protein in pan-cancer. We investigated the expression of NEK2 protein in normal and tumor tissues of different organs of the human body by HPA database and showed IHC images of normal and tumor tissues of lymph nodes, cervix, stomach, and uroepithelium (Supplementary Figure S1).
3. 2 The correlation between NEK2 expression and adverse consequences of cancer
To further determine the prognostic value of NEK2, we performed survival analysis on data retrieved from the TCGA database to observe the correlation of NEK2 with OS, DSS, DFI and PFI in different cancers. The Cox proportional-hazards model demonstrated that high expression of NEK2 mRNA was correlated with OS in ACC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, MESO, PAAD, PCPG and UVM, and was negatively correlated with the OS of THYM (Fig. 2A). KM survival analysis was used to evaluate the relationship between NEK2 expression and clinical outcomes. In patients with ACC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, MESO, PAAD, PCPG and UVM, high NEK2 expression was associated with poorer OS, whereas in patients with THYM, high NEK2 expression was associated with better OS (Fig. 2B-L).
In addition, high NEK2 expression correlated with poorer DSS in patients with ACC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, MESO, PAAD, PCPG, PRAD and UVM (Supplementary Figure S2); KIRP, LIHC, LUAD, PAAD, PRAD, SARC, THCA patients correlated with poor DFI (Supplementary Figure S3); high NEK2 expression in ACC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, MESO, PAAD, PCPG, PRAD, SARC, THCA, and UVM patients correlated with poor PFI (Supplementary Figure S3); high NEK2 expression in ACC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, MESO, PAAD, PCPG, PRAD, SARC, THCA, and UVM patients correlated with poorer PFI (Supplementary Figure S4).
3. 3 Mutations in NEK2
Mutations in NEK2 expression in cancer were analyzed by the cBioPortal online tool. Including all TCGA pan-cancer studies, a total of 32 studies and 10,967 samples, and we identified 88 mutation sites between amino acids 0 and 445, including 72 Missense, 9 Truncating, 6 Splice, and 1 Fusion, with R337CH as the most common mutation site (Fig. 3A). NEK2 mutations were most common in BRCA, UCEC, LIHC, CHOL and SKCM, and the predominant mutation types were Missense mutation, Amplification and Deep Deletion (Fig. 3B). Among the 32 cancers, there was a SHALLOW deletion in NEK2 mRNA expression in all cancers except LAML, THYM and UVM (Fig. 3C).
3. 4 Correlations between NEK2 expression and immune checkpoints
Immune checkpoint inhibitors (ICI) therapy works by blocking immunosuppressive checkpoints such as PD-1, CTLA-4, and TIGIT in order to activate immunostimulatory checkpoints such as CD226 and CD28 in effector T cells and myeloid cells[32], and this treatment has revolutionized the landscape of malignant tumors. Tumor mutational burden (TMB) is defined as the total number of base mutations per million cells in a tumor. TMB reflects the number of cancer mutations, which can stimulate the production of tumor-specific and highly immunogenic antibodies, and is a new target for predicting the efficacy of tumor immunotherapy[33]. Microsatellite instability (MSI) refers to any change in microsatellite length of a microsatellite due to insertion or deletion of repetitive units in a microsatellite in a tumor compared to normal tissues, and the phenomenon of new microsatellite alleles, which leads to impaired gene replication and tumor progression, and affects the prognosis of the tumor[34]. TMB and MSI are relevant biomarkers for ICI[35]. We investigated the correlation between NEK2 expression and TMB and MSI in all TCGA cancers. NEK2 was found in ACC, BLCA, BRCA, COAD, HNSC, KICH, KIRC, LAML, LGG, LUAD, LUSC, MESO, PAAD, PRAD, READ, SARC, SKCM, STAD, Expression in TGCT, THCA, and UCEC was positively correlated with TMB and negatively correlated with TMB in THYM (Fig. 4A). A significant positive correlation of NEK2 with MSI was observed in BLCA, COAD, ESCA, LIHC, MESO, READ, SARC, STAD, and UCEC (Fig. 4B).
Next, we extracted a total of 11 immune checkpoint genes (including PDCD1, CTLA4, C10orf54, HAVCR2, LAG3, TIGIT, SIRPA, BTLA, SIGLEC7, LILRB2, and LILRB4 from the TCGA dataset for the immune checkpoint gene correlation analysis. The results showed that most immune checkpoint genes were positively correlated with NEK2 in all tumor types (Fig. 4C).
3. 5 Correlations between NEK2 expression and immune infiltration
An increasing number of reports suggest that the tumor immune microenvironment plays a crucial role in tumorigenesis and progression[36]. Therefore, we further explored the pan-cancer relationship between the tumor microenvironment and NEK2 expression. The ESTIMATE algorithm was used to calculate stroma and immune cell scores for 33 cancers and analyse the relationship between NEK2 expression levels and the two scores. The stromal score reflected the proportion of stromal cells in tumor tissues; the immune score reflected the proportion of infiltrating immune cells in tumor tissues. The results showed a significant negative correlation between NEK2 expression and stromal score in BRCA, CESC, COAD, GBM, HNSC, LIHC, LUAD, LUSC, OV, PAAD, PRAD, SKCM, STAD, TGCT, THYM and UCEC. This suggests that in these types of tumours, high expression of NEK2 is associated with infiltration of stromal cells, leading to high tumour purity, whereas it is positively correlated in KIRC and THCA (Supplementary Fig. S5).In CESC, COAD, ESCA, GBM, LUAD, LUSC, OV, PAAD, SARC, SKCM, STAD, TGCT, and UCEC, NEK2 expression was significantly negatively correlated with immunological scores. This suggests that high NEK2 expression is associated with reduced immune cell infiltration in these tumours, resulting in high tumour purity, whereas a positive correlation was observed in KIRC and THCA (Supplementary Fig. S6).
Next, we investigated the relationship between NEK2 expression levels and the level of infiltration of 39 immune-related cells. The results showed that for most cancers, the immune cell infiltration level was significantly negatively correlated with NEK2 expression (Fig. 5). Among them, NEK2 expression was significantly positively correlated with the immune-related cell infiltration level of Common lymphoid progenitor in 29 cancer types, and T cell CD4 + Th2 in 31 cancer types. The expression level of NEK2 was positively correlated with THYM B cell, CD4 + T cell, and CD8 + T cell.
In addition, co-expression analysis was performed in 33 tumors to detect the relationship between NEK2 expression and immune-related genes. As visualized in the heatmap (Supplementary Figure S7), almost all immune-related genes were co-expressed with NEK2, and most immune-related genes were positively correlated with NEK2 in all types of tumors.
3. 6 The expression pattern of NEK2 at single-cell levels
Single-cell transcriptome sequencing is an important method for studying different types of cancer, immune cells, endothelial cells and stromal cells[37].Next, we explored the relationship between NEK2 expression and different functional states of tumors using the CancerSEA tool, which allowed us to analyse the correlation between NEK2 and multiple functional states of 16 cancers at the single-cell level. NEK2 was positively correlated with cellcycle, invasion, and proliferation in most cancers and negatively correlated with angiogenesis, apoptosis, inflammation, and quiescence (Supplementary Figure S8), which is consistent with previous studies linking NEK2 expression with tumor functional status[12].The results showed a positive correlation between NEK2 expression and cellcycle, invasion and proliferation, and a negative correlation between NEK2 expression and angiogenesis, apoptosis, inflammation and quiescence (Supplementary Figure S8 ). We then explored the correlation between NEK2 and specific tumor functional states. The results showed that NEK2 was positively correlated with proliferation, cellcycle, invasion and EMT and negatively correlated with inflammation and hypoxia in Acute myeloid leukemia (AML). NEK2 was positively associated with cell cycle, proliferation and DNA damage and DNA repair in colorectal cancer (CRC) and LUAD. NEK2 was negatively correlated with DNA repair, DNA damage, apoptosis, EMT and metastasis in uveal melanoma (UM) (Supplementary Figure S9). All of the above data suggest that NEK2 plays an important role in the biological processes of tumorigenesis and progression.
3. 7 PPI network and GO and KEGG enrichment analysis of NEK2 and related genes
To further elucidate the biological function of NEK2 in tumors, the 100 most relevant genes for NEK2 were obtained from the GEPIA2 database (Supplementary Table S1). These 100 NEK2-associated genes were generated as a PPI network on the STRING website (Fig. 6A). Figure 6 shows the results of GO analysis and KEGG pathway analysis, which includes three categories: biological pathways (BP), cellular components (CC) and molecular functions (MF). GO analysis (Fig. 6B) indicated that NEK2-related genes may be involved in biological pathways such as“organelle fission”, “nuclear division”, “mitotic nuclear division”and“chromosome segregation".NEK2-related genes may be involved in cellular components such as “spindle”,“chromosomal region ”,“chromosome, centromeric region”and“condensed chromosome, centromeric region”. NEK2-related genes may be involved in molecular functions such as “tubulin binding”,“microtubule binding”,“cytoskeletal motor activity”and“microtubule motor activity”. KEGG pathway analysis (Fig. 6C) indicated that NEK2-associated genes may be associated with "Cell cycle", "Oocyte meiosis", " Progesterone-mediated oocyte maturation", "Cellular senescence" and "p53 signalling pathway".
Subsequently, we applied GSEA to determine the biological function of NEK2 in tumors. NEK2 is significantly associated with cell cycle signalling pathways,such as Cell Cycle Checkpoints and Mitotic Spindle Checkpoints. These results suggest a molecular mechanism of NEK2 in tumorigenesis(Supplementary Figure S10).
3. 8 Analysis of drug sensitivity to the NEK2 gene
Finally, we used the CellMiner database to elucidate the potential correlation between NEK2 expression and pan-cancer drug sensitivity. NEK2 expression was positively correlated with the drug sensitivity of B-7100, BMS-754807, CCT-128930, Dexrazoxane, ENMD-2076, Epothilone B, Imiquimod, LEE- 011, LY-2835219, maritoclax, Nitrogen mustard and VT-464 were positively correlated with drug sensitivity(Fig. 7) .
3.9 NEK2 regulates the proliferation of cervical cancer cells
To determine the expression levels of NEK2 in tumor and normal cell lines, immunohistochemical, Western blot and RT-qPCR analyses were performed. The results showed that the expression level of NEK2 in cervical cancer tissues and cell lines was higher than that in normal cells (Figs. 8A, 8B, 8C). Based on these results, the function of NEK2 in Hela and Siha cell lines was further investigated. CCK-8 assay showed that cell proliferation was inhibited in the JH295 group compared to the control group (Fig. 8D). Therefore, these results suggest that NEK2 is highly expressed in cervical cancer and promotes the proliferation of cervical cancer cells.