Kinetochore-associated protein 1 as a prognostic biomarker in gastric cancer: Integrative bioinformatics and experimental validation

Figure 1 Kinetochore-associated protein 1 expression in pan-cancer and gastric cancer tissues. A: Kinetochore-associated protein 1 (KNTC1) expression in pan-cancer tissue from The Cancer Genome Atlas and Genotype-Tissue Expression database; B and C: KNTC1 expressions in tumor and unpaired normal tissues (B) and paired adjacent normal tissues (C) from The Cancer Genome Atlas gastric cancer (GC) database; D and E: Immunohistochemical staining of KNTC1 in GC tissues and adjacent nontumorous tissues, with a representative image from five paired samples (scale bar = 100 μm). Immunohistochemical scoring was performed using the H-score system, where the staining intensity (IS) was graded from 0 to 3, and the percentage of positively stained tumor cells (AP) was graded from 0 to 4. The H-score was calculated as ∑(IS × AP); F: Expression of KNTC1 in GC was assessed through immunohistochemistry and western blotting; G: Expression of KNTC1 in GSE65801 (n = 32, T = 32) database; H: Area under the curve of KNTC1 in GC. ACC: Adrenocortical carcinoma; BLCA: Bladder urothelial carcinoma; BRCA: Breast invasive carcinoma; CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: Cholangiocarcinoma; COAD: Colon adenocarcinoma; DLBC: Lymphoid neoplasm diffuse large B-cell lymphoma; ESCA: Esophageal carcinoma; GBM: Glioblastoma multiforme; HNSC: Head and neck squamous cell carcinoma; KICH: Kidney chromophobe; KIRC: Kidney renal clear cell carcinoma; KIRP: Kidney renal papillary cell carcinoma; LAML: Acute myeloid leukemia; LGG: Brain lower grade glioma; LIHC: Liver hepatocellular carcinoma; LUAD: Lung adenocarcinoma; LUSC: Lung squamous cell carcinoma; MESO: Mesothelioma; OV: Ovarian serous cystadenocarcinoma; PAAD: Pancreatic adenocarcinoma; PCPG: Pheochromocytoma and paraganglioma; PRAD: Prostate adenocarcinoma; READ: Rectum adenocarcinoma; SARC: Sarcoma; SKCM: Skin cutaneous melanoma; STAD: Stomach adenocarcinoma; TGCT: Testicular germ cell tumors; THCA: Thyroid carcinoma; THYM: Thymoma; UCEC: Uterine corpus endometrial carcinoma; UCS: Uterine carcinosarcoma; UVM: Uveal melanoma; KNTC1: Kinetochore-associated protein 1; AUC: Area under the curve; CI: Confidence interval.

Figure 2 Expression levels of kinetochore-associated protein 1 among gastric cancer patients with different clinical characteristics. A: Gender; B: T classification; C: M classification; D: N classification; E: Histological grade; F: Pathologic stage; G: Helicobacter pylori infection. KNTC1: Kinetochore-associated protein 1.

Figure 3 Elevated kinetochore-associated protein 1 expression is associated with worse outcomes. A-C: Kaplan-Meier survival curves showing overall survival, progression-free survival and post-progression survival based on varying kinetochore-associated protein 1 expression levels; D-F: Forest plots summarized the association between kinetochore-associated protein 1 expression and prognosis across different clinical parameters. OS: Overall survival; FP: Progression-free; PPS: Post-progression survival; KNTC1: Kinetochore-associated protein 1; HR: Hazard ratio; CI: Confidence interval.

Figure 4 Gene network associated with kinetochore-associated protein 1. A: Protein-protein interaction networks of kinetochore-associated protein 1 (KNTC1)-interacting proteins were generated using the STRING database; B-D: Association between KNTC1 and the top three KNTC1-correlated genes (POLE, NUP205 and SFPQ) from The Cancer genome Atlas database; E: Intersection analysis of KNTC1-interacting and correlated genes; F: Heatmap illustrating the correlations between KNTC1 and relative genes across various tumors. KNTC1: Kinetochore-associated protein 1.

Figure 5 Comprehensive bioinformatic analyses of kinetochore-associated protein 1 in gastric cancer. A-D: Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of the top 100 kinetochore-associated protein 1 (KNTC1)-associated genes; E: Heat maps showing the association of KNTC1 and top 30 related genes in gastric cancer; F: CBioPortal OncoPrint graph illustrating KNTC1 genomic alterations in gastric cancer patients.

Figure 6 Correlation between kinetochore-associated protein 1 expression and immune cell infiltration in gastric cancer. A: Scatter plot of kinetochore-associated protein 1 (KNTC1) expression and immune cells infiltration in gastric cancer from TIMER database; B: Infiltration levels of several immune cell types in KNTC1 high or low expressed gastric cancer tissues; C: Spearman’s method revealing the correlation between immune cell levels and KNTC1 expression.

Figure 7 Investigating the correlation between cancer-associated fibroblasts and kinetochore-associated protein 1 expression among various cancer types in The Cancer Genome Atlas. A: Partial correlation heatmap of kinetochore-associated protein 1 expression and cancer-associated fibroblast infiltration levels across multiple cancer types; B-I: Scatterplots.

Figure 8 The forest plot and Kaplan-Meier plot summarizes the prognostic value of kinetochore-associated protein 1 expression in gastric cancer patients with various immune cell subsets. A: Forest plot; B: Kaplan-Meier plot. KNTC1: Kinetochore-associated protein 1; HR: Hazard ratio; CI: Confidence interval.

Figure 9 Kinetochore-associated protein 1 knockdown inhibited gastric cancer development in vitro and in vivo. A and B: Kinetochore-associated protein 1 expression of gastric cancer cells and the knockdown efficiency of short hairpin RNA (shRNA) were detected; C-I: The colony formation, wound-healing, and Transwell assays were used in both control and sh-kinetochore-associated protein 1 NUGC3 groups, with the corresponding statistical analyses presented; J: Expression of cyclin-dependent kinase 4, cyclin-dependent kinase 6 and cyclin D1 was analyzed by western blotting; K: The scheme of xenograft tumor and corresponding tumor volume was measured; L and M: Tumor images were captured, and tumor weights were measured post-sacrifice in the mice. Data are presented as mean ± SD (n = 3 or 6, depending on the experiment). KNTC1: Kinetochore-associated protein 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; CDK: Cyclin-dependent kinase.