Abnormal Expression Level of NCAPH in Pan-Cancers and LUAD
By using Wilcoxon rank sum test, pan-cancer analysis was conduct to compare NCAPH levels in tumor samples of GTEx combined with TCGA and matched normal samples. NCAPH abnormally expressed in bladder urothelial carcinoma (BLCA), Breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma(ESCA), head and neck squamous cell carcinoma (HNSC), renal chromophobe cell carcinoma (KICH), renal clear cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma(LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma(LUSC), pancreatic cancer (PAAD), prostate cancer (PRAD), rectum adenocarcinoma(READ), gastric cancer (STAD), thyroid cancer (THCA), endometrial cancer (UCEC) (P < 0.05)(Figure 1A and Figure 1B). NCAPH levels in 59 normal samples and 535 LUAD samples were compared in TCGA LUAD dataset. In LUAD samples, NCAPH expression was substantially increase (P < 0.001) (Figure 1C). Furthermore, NCAPH expression in 57 LUAD samples and matched normal samples were significantly different (P<0.001) (Figure 1D). According to these findings, we used QPCR and western blotting to quantity NCAPH level on 20 paired LUAD samples and matched normal samples. QPCR and western blotting results showed NCAPH expression elevated in LUAD tissues (P<0.05) (Figure 1E and Figure 1F). Furthermore, receiver operating characteristic (ROC) was used to evaluate diagnostic value of NCAPH between LUAD and normal lung tissue. The area under the curve (AUC) of NCAPH was 0.967(Figure 1G). These results meant NCAPH might be a potentially good diagnostic maker for LUAD.
Identification of NCAPH associated DEGs in LUAD
DEGs analysis involved 267 LUAD NCAPH-high samples and 268 NCAPH-low samples (control group). 1592 DEGs were identified and included 1167 upregulated genes and 425 downregulated genes (adjusted P-value<0.05, Log2-fold change>1.5) (Figure 1H). Then, DEGs in HTSeq-Counts were further analyzed by DESeq2 package. The genes of top 20 DEGs in the between two groups were presented in Figure 1I.
Function Enrichment and Analysis of NCAPH Related Genes in LUAD
To further study functional enrichment information of NCAPH related genes, Metascape was utilized for GO enrichment analysis. NCAPH-related genes play roles in various biological processes. (BPs), cellular compositions (CCs) and molecular functions (MFs), including distal axon, neuron projection terminus, axon terminus, integrator complex, pre-miRNA processing, RNA 3'−end processing and miRNA catabolic process (Figure 2A).
Potential Mechanism of NCAPH in Progression of LUAD
To explore roles of NCAPH in LUAD pathway, we utilized GSEA to analyze differences between NCAPH-high and -low cohorts (adjusted P<0.05, FDR P value<0.25) (c2.cp.biocarta and hall. v6.1 symbols). According to normalized enrichment score (NES), top significant enriched pathways associated with NCAPH high expression were selected. G2M checkpoint, ncRNA metabolic process, memory B cells, KRAS signaling, E2F targets and MIER1 process were significantly enriched in patients with NCAPH (Figure 2B-G).
Correlation Between NCAPH Expression and Immune Infiltration.
We further analyzed the correlation between NCPAH expression and immune cell infiltrating by ssGSEA and spearman correlation. The relationship between NCAPH expression and immune cell infiltration was quantified by ssGSEA and analyzed by spearman correlation. The expression of NCAPH was positively associated with the level of acquired immunocyte [Th2 cells (R = 0.790, P< 0.001)] and negatively correlated with the abundance of innate immunocyte [Mast cell (R = -0.510, P< 0.001)] (Figures 3A-E).
The Correlation Between Expressions of NCAPH and Clinicopathologic Characteristics
513 patients’ characteristics and NCAPH expression data were collected from TCGA to explore the relationship between NCAPH expression and clinicopathologic perimeters. Table 1 and Figure 4A-F shown high expression of NCAPH was significantly related to T stage (T1&T2/T3&T4 vs normal, P < 0.001), N stage (N0&N1/N2&N3 vs normal, P < 0.001), M stage (M1&M0 vs normal, P < 0.001), pathologic stage (stages III&IV vs. stage I, P < 0.001), primary therapy outcome (CR&PR vs normal, P < 0.001) and smoker (Yes vs normal, P < 0.001). Furthermore, we conducted univariate analysis to investigate NCAPH expression whether a dependent variable is associated with poor prognostic clinicopathological characteristics (Table 2). Higher expression of NCAPH in LUAD was positively correlated to T stage (OR = 1.932 for T2&T3&T4 vs. T1), pathologic stage (OR = 1.574 for Stage III& Stage IV vs. Stage I & Stage II), gender (OR = 1.671 for Male vs. Female), number of packs smoked per year (OR = 1.7566 for Mild and Severe vs. None), vascular invasion (OR = 1.05 for yes vs. no), race (OR = 1.07 for ≥40 vs. <40) and smoker (OR = 2.566 for Yes vs. No) (all P<0.05). High expression of NCAPH in LUAD was negatively correlated to primary therapy outcome (OR = 0.562 for PR&CR vs. PD&SD, P = 0.012).
High NCAPH expression Associated with Poor Prognosis of LUAD patients.
A univariate logistic regression was used to explore NCAPH role in LUAD patient prognosis. OS of LUAD NCAPH-high expression was significantly shorter (HR = 1.92, 95%Cl:1.37-2.70). In addition, PFI and DSS in NCAPH high expression group were significantly lower in compared with NCAPH low expression (Figure 5A-H). We also performed subgroup analysis of prognosis. Subgroup analysis results shown the survival of high NCAPH level was poor in T1&T2, T3&T4, N0&N1, M0 and M1 group. In order to further evaluate NCAPH role in LUAD prognosis, multivariate regression was applied with T stage, N stage, M stage, pathologic stage, primary therapy outcome, gender and smoker. In multivariate analysis, high NCAPH expression was still an independent prognostic factor (Table 3).
NCAPH--related prognostic nomogram
To predict the prognosis value of NCAPH in LUAD, we established a nomogram and risk classification for predicting 1 year survival (Figure 6A). According to clinical relevance and multivariate Cox analysis results, variables in nomogram were selected. With the adjusted range from 1 to 100, points of each variable were added up and total scores were calculated. By delineating a direct line down from the total score line to the outcome line, the probable prognosis of each LUAD patients at 1years were defined. For example, a LUAD patient with high NCAPH risk (56 points), T3&T4(98 points), N2&N3(100 points), primary therapy outcome (100 points), smoker (30 points) account for 384 points. The probability of 1 year survival was about 56% (Figure 6A). The efficacy of the nomogram were also evaluated, and the result showed that the ability of prediction efficiency of the nomogram was moderately accurate (Figure 6B).
Knockdown of NCAPH suppress malignant phenotype of lung adenocarcinoma in vitro
H2122 and H3122 cell line were chosen to research the role of NCAPH in LUAD. Three NCAPH siRNAs were transfected into cells. NCAPH mRNA expressions were tested to evaluate three NCAPH siRNAs knockdown efficiency. Among these siRNAs, siRNA showed most significant inhibition ratio and was selected for further experiment. MTT assay data indicated that siRNA targeting NCAPH significantly reduced cell growth rates. Transwell assay revealed that NCAPH-targeted siRNAs transfection notably reduced migration and invasion in both cell lines. By using flow-cytometry analysis, the cell-cycle distribution of NCAPH siRNA transfected cells was increasing in G1/G0 cell population in both cell lines. Besides, the apoptosis of H2122 and H3122 cells remarkable increased in NCAPH siRNA treatment group, by using the Annexin V-FITC/PI double staining technique. These data were showed in Figure 7 and Figure 8.