3.1 Expression level of NCAPD2 mRNA expression in clinical
The RNA sequence of 33 cancers from TIMER2.0 database analyzed that NCAPD2 mRNA level was considerably increased in 16 cancers compared with the normal tissue, including HCC, LUAD, and BRCA, while its expression was down-regulated in SKCM and THCA (Fig. 1A). To provide a comprehensive characteristic of NCAPD2 expression, we analyzed the potential relationship between NCAPD2 and the stage of pan-cancer. NCAPD2 showed significant association with the pathological stage of ACC, BRCA, KICH, KIRC, KIRP, LIHC, LUAD, SKCM, TGCT (Fig. 1B). Meanwhile, data indicated that the association of NCAPD2 expression and gender in multiple cancers, the significant difference existence in HNSC, LAML, LIHC, LUAD, LUSC and SARC (Fig. 1C). To evaluate the expression level of NCAPD2 in HCC, the analysis revealed that NCAPD2 mRNA level were significantly higher in HCC than normal tissues (Fig. 1D), and compared with adjacent normal tissues (Fig. 1E) (paired samples t-test).
3.2 Survival analysis
To estimate the prognostic value of NCAPD2 in tumor, the Kaplan-Meier curve assisted in depicting the correlation between the abnormal level of NCAPD2 and the pan-cancer survival based on the TCGA database. The results revealed that the survival rate of patients with high expression level of NCAPD2 in LIHC (P = 0.028), LUAD (P = 0.028), UCEC (P = 0.033) and KIRP (P <0.0001) (Figs. 2A–D). For further comprehensive evaluation of the specific effect of NCAPD2 on the survival, the assessment of abnormal NCAPD2 level on the disease-specific survival (DSS) and progression free survival (PFS) of tumor patients was depicted. The Kaplan-Meier plots showed that the increased expression of NCAPD2 was along with poor DSS and PFS in various cancers (Figs. 2E–L).
3.3 NCAPD2 expression is related to methylation, tumor mutational burden and microsatellite instability in human cancers
DNA methylation is the production of 5-methycytosine by adding a methyl group to the fifth carbon atom of cytosine in DNA sequence after the DNMT catalysis. The specific gene methylation could exact various biological roles in cancers, like as the marker for drug efficacy and involving in the occurrence and progression of cancers24. Besides, due to the significant association between DNA methylation and immune response in different cancers, we investigated the methylation of NCAPD2 in pan-cancer. As shown in the Fig. 3A-J, the methylation level of NCAPD2 was negative correlative in LIHC, LUAD, UCEC, CESC, ACC, COAD, LUSC, SKCM, STAD, UCS compared with their cancer tissue. DNA methylation analysis of NCAPD2 could provide a novel sight of the prognosis of NCAPD2 in cancer treatment. Additionally, previous researches found that tumor mutation load (TMB) and microsatellite instability (MSI) are the crucial factors that effect tumor genesis and development[25, 26]. Herein, the association between MSI or TMB and NCAPD2 expression was analyzed in multiple types of cancers. Data presented that NCAPD2 showed substantial positive association with TMB in ACC, BLCA, BRCA, COAD, HNSC, KICH, LGG, LUAD, LUSC, PAAD, PRAD, READ, SARC, SKCM, STAD, TGCT, UCEC (Fig. 3K), and negative correlated in ESCA, THCA, THYM. Besides, results showed that the NCAPD2 presented significant relation with MSI in UCEC, STAD, SARC and COAD (Fig. 3L).
3.4 Correlation analysis of NCAPD2 in hepatocellular carcinoma
According to the analysis of gene expression, survival analysis, mutation analysis and the methylation level of NCAPD2 in pan-cancer, which also indicated that NCAPD2 may exact a key role in HCC development. Subsequently, to evaluate the clinical analysis of NCAPD2, the correlation of the NCAPD2 expression and several clinical indicators were assessed in HCC based on the TCGA data. The results about the pathological stage showed that NCAPD2 expression was significant upregulated in HCC samples from stage III–IV by comparison with the stage I–II (Fig. 4A). Regarding to the T stage and histologic grade, the results suggested that higher expression of the NCAPD2 were presented in the T3 + T4, G3 + G4 by compared with the T1 + T2, G1 + G2, respectively (Fig. 4B, C). Additionally, NCAPD2 expression in female was higher than male, and the expression of NCAPD2 in HCC patients with the age < = 65 was much lower compared with patients with the age > 65 (Fig. 4D, E). However, there was no effect on the distant metastasis and lymphatic metastasis with the overexpressed NCAPD2 (Fig. 4F, G). These findings indicated that upregulation of NCAPD2 was substantially correlation with unfavorable clinicopathological results, including T stage, pathologic stage, age and gender, which proved the positively correlation between NCAPD2 expression and HCC malignant characteristics. To confirm whether NCAPD2 could as the prognostic evaluation marker in HCC, receiver operating characteristics (ROC) curve showed that it had efficacious prognostic evaluation performance, with AUC of 0.726, 0.620 and 0.566 at 1, 3 and 5 years, respectively (Fig. 4H). The univariate regression analysis indicated that NCAPD2 as an independent prognostic mediator was valid in HCC patients (Fig. 4I).
3.5 Functional Enrichment Analysis of DEGs Between HCC with NCAPD2 Expression
The above analysis showed that NCAPD2 played a significant clinical diagnostic and prognostic value of HCC, and discovered that its downregulation was association with poor outcomes. The OPENTARGET platform was further to analyze the related diseases network about the NCAPD2. The high expression of NCAPD2 was present substantial positive association with nervous system diseases, cancer or benign tumor, gastrointestinal disease and disorder of visual system, genetic familiar or congenital disease (Fig. 5A). To deeply study the molecule mechanism of NCAPD2 in tumorigenesis, GeneMANIA was used to predict the binding protein of NCAPD2. As showed in Fig. 5B, NCAPD2 has potential interactions with NCAPG, SMC4, NCAPH, SMC2 and NCAPD3, which exacted main function, like chromosome segregation, condensed chromosome and mitotic nuclear division and so on. Subsequently, GO terms and KEGG pathway were performed to analyze the relative molecule function that NCAPD2 referred to. Biological processes enrichment results suggested that NCAPD2 may be associated with nuclear division, B cell activation and B cell receptor pathway. Cellular component enrichment analysis showed that NCAPD2 could exact a significant role in immunoglobulin complex and T cell receptor complex, and molecule function enrichment analysis showed that NCAPD2 played an important role in channel activity and passive transmembrane transporter activity. Enrichment analysis of biological process results gave a hint that these molecule may involved in ion channel activity and immunoglobulin receptor binding (Fig. 5C-5D). Meanwhile, KEGG analysis indicated that the top enrichment terms related to NCAPD2, including Neuroactive ligand − receptor interaction, cAMP signaling pathway, cell cycle and cell adhesion, which may be contributed to the poor prognostic of HCC patients (Fig. 5D). Besides, Single-sample gene set enrichment analysis (ssGSEA) algorithm was introduced to speculate the infiltration levels of immune cells types about NCAPD2 in HCC. The analysis showed that NCAPD2 expression was positive correlation with T helper cells, CD4 + T cells memory, and negative correlation with CD4- T cells and Macrophages M1 (Fig. 5E). Meanwhile, increased studies have confirmed that immune checkpoints (ICPs) exact important roles in tumor infiltration and immunotherapy27. Thus, we analyzed the immune checkpoint inhibitors referred to NCAPD2 expression, which showed bound up of the expression NCAPD2 was significant positively association with TNFSF18, TNFSF15, TIGIT and CD276, was strong negatively correlated with TNFRSF25, CD200, LAG3 and CD244 in HCC. The above results suggested that NCAPD2 might be an ideal tumor immunotherapeutic target and play certain roles in tumor immunotherapy response and outcome (Fig. 5F) .
3.6 The expression of NCAPD2 is crucial for the Immune cell infiltration
In view of the results that the GO and KEGG analysis of NCAPD2 and its co-expressed gene in liver cancer also showed the obvious association with T and B cells in immunity (Fig. 5C-D). Besides, increased studies have confirmed that immune checkpoints (ICPs) exact important roles in tumor infiltration and immunotherapy[27]. To discuss the potential role of NCAPD2 in immune cell infiltration from the TIMER2.0 database, the following outcomes demonstrated that high levels of MDSC cells, CD8 + central memory cells, Hematopoietic stem cells and Macrophages M0 cells were correlation with poor prognosis in HCC patients and high NCAPD2 level (p < 0.05) (Fig. 6A, B, C, D). Macrophages M2 cells and Macrophage cells infiltration were significantly associated with poor prognosis in patients in the low NCAPD2 expression group (p < 0.05) (Fig. 6E and F). These findings suggested that NCAPD2 had an important role in the immune infiltration. To identify NCAPD2 as immunotherapeutic target may help to improve the survival of patients[28, 29]. Previous studies showed that combination therapy with PD1 and CTLA4 inhibitor could result in improving the cancer patients survival[30–33]. In this study, data suggested that high expression of NCAPD2 exerted the significant role in immunotherapy effect was under the PD1 and CTLA4 positive, indicated that suppression of theses molecules represented a new method for the immune checkpoint blockade therapy (Fig. 7A-D). Besides, the association between drug sensitivity (IC50) and NCAPD2 expression were analyzed. The results suggested that the high expression group of NCAPD2 was showed the lower IC50 of HCC clinical therapeutics, including sorafenib, 5-Fluorouracil, Paclitaxel, Gemcitabine (Fig. 7E-H), which suggested the sensitive of NCAPD2 expression with the HCC therapeutics.
3.7 The effect of NCAPD2 on the proliferation, migration, invasion, cell cycle and apoptosis of hepatocellular carcinoma cells
To confirm the role of NCAPD2 in HCC, immunohistochemical staining showed that NCAPD2 expression level was much higher in tumor tissues compared with paracerous ones from HPA database (Fig. 8A). Subsequently, RT-qPCR and western blotting were conducted to test its expression in normal hepatocyte cell line THLE-2 along with the other HCC cells (Huh-7, SK-Hep-1 and HepG2). The results demonstrated that the level of NCAPD2 were higher in hepatocellular carcinoma cells than the normal cells (Fig. 8B-C). Based on the oncogenic role of NCAPD2, there was a speculation that NCAPD2 could aggravated the progression of HCC. Thus, NCAPD2 siRNA was constructed and transfected into SK-Hep-1 and HepG2 cells, RT-qPCR and western blotting were performed to confirm the efficiency of NCAPD2 silence (Fig. 8D-E). Subsequently, BrdU assay was carried out to evaluate the effect on proliferation in NCAPD2 knockdown cells. As expected, the results suggested that NCAPD2 knockdown significantly suppressed the proliferation in HCC cells (Fig. 8F-G). Meanwhile, Transwell assay were conducted to evaluate the effect of NCAPD2 knockdown on migration and invasion capacity, and siNCAPD#1 and siNCAPD#2 remarkably suppressed the migration and invasion capacity in SK-Hep-1 and HepG2 cells (Fig. 9A-D), which accorded with the KEGG enrichment analysis that NCAPD2 exerted an important role in cell adhesion.
To further investigate the effect of NCAPD2 on cell cycle, flow cytometer data showed that knock down NCAPD2 accumulated the G0/G1 phase cells (Fig. 10A-C), that confirmed to the analysis of KEGG. Moreover, due to the apoptosis correlation with the retardation of tumor, there was a hypothesis that NCAPD2 silence inhibited the growth of HCC by apoptosis occurrence. The flow cytometry result verified the assumption that abrogation of NCAPD2 could enhance apoptosis activity in HCC cells (Fig. 10D-F). Collectively, NCAPD2 as an oncogene, aggravated the HCC progression, and silencing NCAPD2 inhibits the activity of various HCC cells. There was be a potential association with EMT procedure, cell cycle and apoptosis to be further elucidate.