1. Relationship between KNSTRN expression and clinical characteristics
KNSTRN expression in LUAD and normal tissues was analyzed, revealing a difference in the KNSTRN expression levels in LUAD and normal tissues; KNSTRN was highly expressed in tumor tissues (P < 0.001, Fig. 1A). At the same time, KNSTRN expression in LUAD tissues and paired adjacent nontumorous tissues were analyzed. The results also suggested that KNSTRN was highly expressed in tumor tissues (P < 0.001, Fig. 1B). KNSTRN expression in tumor tissues was standardized using the Z-score, and the LUAD cohort was divided into high- and low-expression groups according to KNSTRN expression (Fig. 1C). In addition, ROC curves were used to analyze the diagnostic value of KNSTRN. The area under the curve (AUC) of KNSTRN was 0.815, and the results suggested that KNSTRN might be a potential diagnostic biomarker (Fig. 1D).
In addition, the Kruskal–Wallis test and Wilcoxon signed-rank test were used to analyze the relationship between KNSTRN expression and clinical characteristics. The increased expression levels of KNSTRN positively correlated with higher grades of T stage (P < 0.001, Fig. 2A), N stage (P = 0.003, Fig. 2B), M stage (P = 0.02, Fig. 2C), clinical stage (P < 0.001, Fig. 2D), tumor status (P < 0.001, Fig. 2E), and the outcome of the primary therapy (P = 0.003, Fig. 2F). At the same time, consistent results were also found using the chi-square test or Fisher exact test (Table 1). Moreover, the univariate logistic regression of KNSTRN expression also suggested a close relationship between KNSTRN and clinical characteristics., including T stage [odds ratio (OR) = 1.04 (1.02–1.07), P < 0.001], N stage [OR = 1.02 (1.01–1.04), P = 0.010], clinical stage [OR = 1.03 (1.01–1.05), P = 0.003)], tumor status [OR = 1.03 (1.01–1.05), P = 0.002], outcome of the primary therapy [OR = 1.04 (1.02–1.07), P < 0.001], and TP53 mutation [OR = 1.04 (1.02–1.06) ), P < 0.001] (Table 2). No significant difference was found in the relationship with M stage [OR = 1.02 (0.99–1.05), P = 0.099]. These results suggested that KNSTRN expression was related to clinical characteristics.
Table 1
Relationship between KNSTRN expression and clinicopathological characteristics in patients with lung adenocarcinoma
Characters | level | Low expression of KNSTRN | High expression of KNSTRN | p | test |
n | | 256 | 257 | | |
OS event (%) | Alived | 180 (70.3) | 146 (56.8) | 0.002 | |
| Dead | 76 (29.7) | 111 (43.2) | | |
T stage (%) | T1 | 104 (40.9) | 64 (25.0) | < 0.001 | exact |
| T2 | 125 (49.2) | 151 (59.0) | | |
| T3 | 21 ( 8.3) | 26 (10.2) | | |
| T4 | 4 ( 1.6) | 15 ( 5.9) | | |
N stage (%) | N0 | 179 (72.2) | 151 (59.7) | 0.002 | exact |
| N1 | 45 (18.1) | 50 (19.8) | | |
| N2 | 23 ( 9.3) | 51 (20.2) | | |
| N3 | 1 ( 0.4) | 1 ( 0.4) | | |
M stage (%) | M0 | 164 (95.9) | 180 (90.9) | 0.090 | |
| M1 | 7 ( 4.1) | 18 ( 9.1) | | |
Clinical stage (%) | Stage I | 156 (62.2) | 118 (46.5) | < 0.001 | |
| Stage II | 60 (23.9) | 61 (24.0) | | |
| Stage III | 27 (10.8) | 57 (22.4) | | |
| Stage IV | 8 ( 3.2) | 18 ( 7.1) | | |
Tumor status (%) | Tumor free | 157 (67.1) | 131 (58.7) | 0.080 | |
| With tumor | 77 (32.9) | 92 (41.3) | | |
Primary therapy outcome (%) | PD | 23 (10.1) | 45 (22.6) | 0.003 | exact |
| SD | 23 (10.1) | 14 ( 7.0) | | |
| PR | 4 ( 1.8) | 2 ( 1.0) | | |
| CR | 177 (78.0) | 138 (69.3) | | |
Age (%) | < 65 | 98 (39.4) | 122 (49.8) | 0.025 | |
| >= 65 | 151 (60.6) | 123 (50.2) | | |
Gender (%) | Female | 158 (61.7) | 118 (45.9) | < 0.001 | |
| Male | 98 (38.3) | 139 (54.1) | | |
Anatomic subdivision (%) | Left | 107 (42.6) | 92 (37.2) | 0.257 | |
| Right | 144 (57.4) | 155 (62.8) | | |
Smoking status (%) | Non-smoker | 46 (18.5) | 28 (11.2) | 0.028 | |
| Smoker | 202 (81.5) | 223 (88.8) | | |
TP53 mutation (%) | No | 165 (65.0) | 102 (40.2) | < 0.001 | |
| Yes | 89 (35.0) | 152 (59.8) | | |
Table 2
Logistic regression analysis of KNSTRN expression
Characteristics | Total (N) | Odds ratio in KNSTRN expression | P value |
T stage (T1 vs. T2-4) | 510 | 1.04(1.02–1.07) | < 0.001 |
N stage (N0 vs. N1-3) | 501 | 1.02(1.01–1.04) | 0.01 |
M stage (M0 vs. M1) | 369 | 1.02(0.99–1.05) | 0.099 |
Clinical stage (Stage I vs. Stage II-IV) | 505 | 1.03(1.01–1.05) | 0.003 |
Tumor status (Tumor free vs. With tumor) | 457 | 1.03(1.01–1.05) | 0.002 |
Primary therapy outcome (CR-SD vs. PD) | 426 | 1.04(1.02–1.07) | < 0.001 |
Anatomic subdivision (Left vs. Right) | 498 | 1.01(0.99–1.02) | 0.547 |
TP53 mutation (No vs. Yes) | 508 | 1.04(1.02–1.06) | < 0.001 |
2. Prognostic value of KNSTRN expression in LUAD
The relationships of KNSTRN expression with prognostic outcomes in overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS) are shown in Fig. 3A–3C. High expression of KNSTRN was associated with poor OS [hazard ratio (HR) = 1.730 (1.288–2.324], P < 0.001, Fig. 3A], poor PFS [HR = 1.397 (1.062–1.838), P = 0.017, Fig. 3B], and poor DSS [HR = 1.967 (1.345–2.875), P < 0.001, Fig. 3C]. At the same time, this study also showed the distribution of high expression and low expression of KNSTRN with respect to OS and risk score (Fig. 3D). The results suggested that patients with LUAD and high risk scores had high expression levels of KNSTRN, while patients with low risk scores were associated with low expression levels of KNSTRN.
In addition, this study analyzed the relationship between KNSTRN expression and different subgroups. KNSTRN was found to be highly expressed in N0 stage [HR = 1.814 (1.183–2.780), P = 0.006], M0 stage [HR = 1.713 (1.201–2.444), P = 0.003], stage I [HR = 1.730 (1.062–2.817), P = 0.028], age > 65 years [HR = 2.104 (1.428–3.098), P < 0.001], and smokers [HR = 1.833 (1.311–2.564), P < 0.001]; high expression of KNSTRN was related to poor OS (Table 3 and Fig. 3E).
Table 3
Prognostic analysis of KNSTRN expression in a subset of patients with LUAD
Characteristics | N (%) | Hazard ratio | P value |
T stage | | | |
T1 | 168 (34) | 1.710 (0.938–3.117) | 0.080 |
T2 | 269 (54) | 1.368 (0.925–2.022) | 0.116 |
T3 | 45 (9) | 2.930 (1.166–7.363) | 0.022 |
N stage | | | |
N0 | 325 (66) | 1.814 (1.183–2.780) | 0.006 |
N1 | 94 (19) | 1.184 (0.687–2.039) | 0.543 |
N2 | 71 (14) | 1.528 (0.757–3.086) | 0.237 |
M stage | | | |
M0 | 335 (93) | 1.713 (1.201–2.444) | 0.003 |
Clinical stage | | | |
Stage I | 270 (54) | 1.730 (1.062–2.817) | 0.028 |
Stage II | 119 (24) | 1.038 (0.606–1.778) | 0.892 |
Stage III | 81 (16) | 1.475 (0.768–2.831) | 0.243 |
Age | | | |
< 65 | 220 (45) | 1.544 (0.963–2.477) | 0.071 |
>= 65 | 274 (55) | 2.104 (1.428–3.098) | < 0.001 |
Gender | | | |
FEMALE | 270 (54) | 1.531 (1.023–2.291) | 0.038 |
MALE | 234 (46) | 1.938 (1.232–3.047) | 0.004 |
Anatomic subdivision | | | |
Left | 194 (40) | 1.886 (1.183–3.008) | 0.008 |
Right | 296 (60) | 1.600 (1.078–2.373) | 0.020 |
Smoking status | | | |
Non-smoker | 71 (14) | 1.219 (0.556–2.673) | 0.620 |
Smoker | 419 (86) | 1.833 (1.311–2.564) | < 0.001 |
Moreover, univariate Cox regression was also performed, and the results suggested that TNM stage, clinical stage, tumor status, primary therapy outcome, and high expression of KNSTRN were associated with poor OS (P < 0.05). Moreover, high expression of KNSTRN was an independent prognostic factor for OS [HR = 1.730 (1.288–2.234), P ༜ 0.001, Table 4] as revealed by multivariate Cox hazard regression analysis. Tumor status, primary therapy outcome, and KNSTRN expression were used to construct a clinical prognostic risk score for LUAD (Fig. 3F). At the same time, the prediction accuracy of the model was assessed using a calibration chart (Fig. 3G). The results suggested that the KNSTRN expression level could better predict the 3-year and 5-year survival of patients. In general, all these results suggested that the KNSTRN expression level correlated with the prognosis of patients with LUAD.
Table 4
Univariate and multivariate Cox proportional hazard analyses of KNSTRN expression
Characteristics | Univariate analysis | Multivariate analysis |
HR(95% CI) | P value | HR(95% CI) | P value |
T stage (T1 vs. T2-4) | 1.668(1.184–2.349) | 0.003 | 1.096(0.641–1.874) | 0.739 |
N stage (N0 vs. N1-3) | 2.606(1.939–3.503) | < 0.001 | 1.495(0.739–3.026) | 0.263 |
M stage (M0 vs. M1) | 2.111(1.232–3.616) | 0.007 | 0.92(0.394–2.15) | 0.848 |
Clinical stage (Stage I vs. Stage II-IV) | 2.975(2.188–4.045) | < 0.001 | 1.033(0.477–2.24) | 0.934 |
Tumor status (Tumor free vs. With tumor) | 6.215(4.261–9.064) | < 0.001 | 5.483(3.244–9.266) | < 0.001 |
Primary therapy outcome (CR-SD vs. PD) | 3.978(2.785–5.682) | < 0.001 | 2.478(1.536–3.997) | < 0.001 |
Age (< 65 vs. >= 65) | 1.172(0.871–1.578) | 0.295 | | |
Gender (FEMALE vs. MALE) | 1.06(0.792–1.418) | 0.694 | | |
Anatomic subdivision (Left vs. Right) | 1.024(0.758–1.383) | 0.878 | | |
Smoking status (Non-smoker vs. Smoker) | 0.887(0.587–1.339) | 0.568 | | |
KNSTRN (Low vs. High) | 1.73(1.288–2.324) | < 0.001 | 1.563(1.014–2.409) | 0.043 |
3. Relationship between KNSTRN expression and whole gene expression profile
The gene expression profiling analysis related to KNSTRN was performed to further explore the biological function of KNSTRN in LUAD. A total of 34 downregulated genes and 486 upregulated genes were considered to be significantly associated with KNSTRN expression (logFC > 2 and Padj < 0.01) (Fig. 4a). Further, the top 30 upregulated genes and top 30 downregulated genes among these abnormally expressed genes were shown in the gene expression heat map (Fig. 4B). In addition, based on KNSTRN expression, GO enrichment analysis was performed using Metascape. The biological functions of the KNSTRN gene are associated mainly with hormone activity, regulation of hormone levels, multi-multicellular organism process, and so forth (Fig. 4C).
4. GSEA analysis of KNSTRN expression
GSEA analysis of the TCGA gene expression data was used to identify functional and biological pathways between low and high expression of KNSTRN. Based on the normalized enrichment scores (NESs), the enrichment signaling pathway most significant in terms of KNSTRN gene expression was selected (Fig. 5 and Table 5). GSEA analysis results showed that the highly expressed KNSTRN phenotype was concentrated mainly in cell cycle checkpoints (A), DNA replication (B), cell cycle (C), mitotic spindle checkpoint (D), G2-M checkpoint 9 (E), and M phase (F).
Table 5
GSEA enrichment analysis results
ID | Set Size | Enrichment Score | NES | P value | P adjust | FDR | Rank | Leading_edge |
REACTOME M PHASE | 352 | 0.590 | 1.941 | 0.001 | 0.020 | 0.016 | 14014 | tags = 56%, list = 27%, signal = 41% |
REACTOME CELL CYCLE CHECKPOINTS | 263 | 0.660 | 2.157 | 0.001 | 0.020 | 0.016 | 10642 | tags = 55%, list = 21%, signal = 44% |
REACTOME G2 M CHECKPOINTS | 145 | 0.668 | 2.100 | 0.001 | 0.020 | 0.016 | 14627 | tags = 75%, list = 28%, signal = 54% |
REACTOME DNA REPLICATION | 125 | 0.679 | 2.102 | 0.001 | 0.020 | 0.016 | 11562 | tags = 67%, list = 22%, signal = 52% |
KEGG CELL CYCLE | 124 | 0.675 | 2.085 | 0.001 | 0.020 | 0.016 | 7088 | tags = 44%, list = 14%, signal = 38% |
REACTOME MITOTIC SPINDLE CHECKPOINT | 106 | 0.674 | 2.064 | 0.001 | 0.020 | 0.016 | 10642 | tags = 55%, list = 21%, signal = 44% |
5. Relationship between KNSTRN expression and immune infiltration
Next, the relationship between KNSTRN expression and 24 different immune cell types was evaluated in LUAD. KNSTRN expression had a close positive relationship with T helper 2 (Th2) cells, Tgd, and NK CD56dim cells, and a close negative relationship with T follicular helper (TFH) cells, mast cells, immature dendritic cells (iDCs), and so forth (Fig. 6A). Further research showed significant differences in the KNSTRN expression level among the infiltrating immune cells, including B cells, CD8 T cells, eosinophils, macrophages, TFH, TH2 cells, NK cells, and so on (Fig. 6B).