High expression of SPAG5 in tumors
We explored the role of SAPG5 in cancers based on the Oncomine database, we found the expression of SPAG5 upregulated in tumor compared with normal tissues of various cancer types, including bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, leukemia, liver cancer, lung cancer, lymphoma, ovarian cancer, sarcoma. Oppositely, SAPG5 downregulated in tumor than normal tissues in few cancer types, including breast cancer, kidney cancer, and leukemia (Figure 1A). Additionally, we also examined the expression of SPAG5 using TIMER2.0. As shown in Figure 1B, SPAG5 increasing in bladder urothelial cancer (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pheochromocytoma and paraganglioma (PCPG), prostate adenocarcinoma (PRAD), and stomach adenocarcinoma (STAD) tumors compared with normal tissues, whereas SPAG5 decreasing in kidney chromophobe (KICH), kidney renal papillary cell carcinoma (KIRP), thyroid carcinoma (THCA), uterine corpus endometrial carcinoma (UCEC) tumors compared with normal tissues. Furthermore, we explored the mRNA expression of SPAG5 in NSCLC based on the TCGA and GTEx data. We found that SPAG5 upregulation in LUAD tissues and significantly expressed in different stages (Figure 1C-D). These results revealed that SPAG5 increasing in LUAD both in microarray data and RNA-seq data, it also indicated SPAG5 might act as an oncogene in LUAD.
Prognostic values of SPAG5 in LUAD
The correlation between expression of SPAG5 and clinical outcome was investigated using GEPIA based on TCGA and GTEx data. As shown in Figure 2A-B, OS curve indicated high expression of SPAG5 associated with poor survival of LUAD (HR=1.6, P=0.0012), whereas the expression of SPAG5 not associated with DFS (HR=13, P=0.096). Using PrognoScan, GSE13213 and GSE31210 datasets, including 117 samples and 204 LUAD samples at different clinical stages, and indicated that high expression of SPAG5 associated with poor survival time (OS HR=1.54, Cox P=0.0025; OS HR=1.67, Cox P=0.057; RFS HR=1.91, Cox P=0.00065) (Figure 2C-E). These results suggested that SPAG5 acted as a potential risk factor for LUAD patients. Moreover, we validated the prognostic potential of SPAG5 using Kaplan-Meier Plotter database based on microarray data. As shown in Figure 2F-H, high expression of SPAG5 indicated the poor survival time of LUAD patients (OS HR=1.68, P=1E-15; FP HR=1.68, P=9.6E-08; PPS HR=1.56, P=0.00058). Our finding suggested the prognostic potential of SPAG5 in LUAD.
SPAG5 upregulation associated with tumor malignant phenotypes
To understand the function and underlying mechanism of SAPG5. We explored the functional state of SPAG5 in LUAD based on single-cell level in CancerSEA database. we found that SPAG5 expression associated with multiple tumor cancers, as shown in Figure 3, acute myelocytic leukemia, LUAD, melanoma, breast cancer, prostate cancer, etc. Of interest, SPAG5 expression positively associated with malignant phenotypes, including cell proliferation, cell cycle, DNA damage and repair, epithelial-mesenchymal transition (EMT), invasion, and stemness, whereas negative related to inflammation with the correlation coefficient>0.3 and P<0.05. These data reminded that SPAG5 expression associated the LUAD tumorigenesis and development.
SPAG5 expression correlated with tumor immune infiltration in LUAD
Emerge evidences indicate the landscape of tumor immune infiltration significant associates with outcome and survival of cancer. Here, we examined the relationship between SPAG5 and tumor immune infiltration in LUAD by TIMER. Our data discovered that SPAG5 expression positively related to B cell, CD4+ cell, CD8+ cell, macrophage M0/M1, whereas negatively correlated with macrophage M2 and DC cell (Figure 4A). Furthermore, we also investigated the correlation between SPAG5 expression and immune checkpoints in LUAD. And the results showed that SPAG5 significantly associated with CD274, CTLA4, GZMB, LAG3, PDCD1, and TIGIT in LUAD (Figure 4B). Our data suggested that SPAG5 exerted a key role in immune pathways of LUAD, and regulated the polarization of macrophage.
PPI establishment and mutation of SPAG5 and its co-expression genes in LUAD
We established a PPI network to analyze the interaction of SPAG5 and other proteins. And the network of SPAG5 along with co-expression proteins was displayed in Figure 5, ten hub genes, including KIF11, DLGAP5, KIF2C, BUB1, BUB1B, CCNB2, CDCA8, AURKB, CDC20, and AURKA, significantly interacted with SPAG5 in LUAD. We further investigated the mutation of SPGAG5 and the ten genes using cBioPortal. And we observed nearly 2.3% (SPAG5), 1.3% (KIF11), 2.7% (DLGAP5), 1.5% (KIF2C), 0.6% (BUB1), 0.9% (BUB1B), 0.6% (CCNB2), 0.8% (CDCA8), 0.7% (AURKB), 1.7% (CDC20), 1.2% (AURKA) of LUAD sample emerged genetic alteration (Figure 6). The main genetic alterations of genes were amplification, deep deletion, and missense mutation.
Function annotation of hub genes in LUAD
We then enriched the function of hub genes using Metascape. And the results suggested that 11 hub genes significantly enriched in mitotic nuclear division, cell division, mitotic sister chromatid segregation, spindle organization, pid auraro B pathway, establishment of chromosome localization, microtubule-based movement (Figure 7A-D). Each MCODE component was enriched and data showed that SPAG5 and its co-expression genes mainly involved in molecular functions, including mitotic nuclear division, resolution of sister chromatid cohesion, and nuclear division (Table 1).
Correlation of SPAG5 expression with PD-L1expression and the clinicopathological characteristics of LUAD
Finally, we observed the expression of SPAG5 in the LUAD tumor tissues, and we found relatively high expression of SPAG5 in advanced stage LUAD samples compared with early stage LUAD samples (Figure 8). Generally, we investigated relationship between the expression of SPAG5 and clinicopathological parameters in 140 LUAD samples (Figure 8), and we found SPAG5 expression associated with gender, clinical stages, tumor size, lymph node metastasis, and PD-L1 expression (Table 2). These results validated the previous predication according to the bioinformatics analyses.