Expression Levels of COL4As in Patients with KIRC
According to the online analysis database Oncomine, we found differential expression of COL4As between kidney carcinoma and adjacent tissues (Fig. 1). Several reported databases confirmed that transcriptional levels of COL4A1 [20–23], COL4A2 [20, 22, 24–26], COL4A3 [20], COL3BP [22, 23] in KIRC was shown a statistically significant compare with normal tissues (Table 1). There are insufficient data of the expressed level of COL4A4, COL4A5, COL4A6 to explore the correlation between KIRC and normal tissues. However, the Ulcan and Gepia online database show an obvious difference in each COL4A gene family numbers' expression level except for COL4A3BP (Fig. 2, Fig. 3). Besides, the COL4A1, COL4A2 has increased significantly while COL4A 3, COL4A4, COL4A5, COL4A6 has decreased (p < 0.001 for all).
Correlation of mRNA expression levels of COL4A family genes and the tumor progression related clinicopathological parameters in KIRC patients.
The patients' individual clinicopathological parameters about cancer stages and tumor grades was explored with Gepia and Ulacn database after an over-expressed level of COL4A family members in KIRC tumors was observed. As we found in Fig. 4, there are remarkable correlations between mRNA expressions of COL4A3 (p < 0.001), COL4A3BP (p < 0.001), COL4A4 (p < 0.001), COL4A6 (p = 0.0434) and patient' pathological stages in Gepia database. However, based on the analysis of Ulcan (Fig. 5), we found a significant statistical correlation between all COL4A family genes and patients' different stages (p < 0.01 for all) except for COL4A3BP, which differential expression only observed in stage 4.
Prognostic value of COL4A family genes in KIRC
Using Gepia database, the prognostic value of COL4A family genes in patients were explored based on the difference of overall survival (OS) and disease-free survival (DFS) between high expression group and low expression group (Fig. 6). The OS of KIRC patients shows a statistical difference among all COL4A family genes (p < 0.05 for all), except for COL4A6. However, most of the COL4A family genes was shown significant correlations with kidney cancer individuals' prognosis, including COL4A3, COL4A3BP, COL4A4, COL4A5 (p < 0.05 for all).
The correlations between genetic mutations in COL4A family numbers and OS, DFS of KIRC patients
The online database cBioPortal was used to analyze the genetic mutations of differentially expressed COL4A family members in KIRC patients. Based on Fig. 7A, the mutation rate of COL4A1, COL4A2, COL4A3, COL4A3BP, COL4A4, COL4A5, COL4A6 genes was 8%, 9%, 9%, 11%, 7%, 9%, and 8% in 512 samples. What's more, the association between genetic mutations and the prognosis of KIRC patients was explored by Kaplan-Meier module and log-rank test in cBioPortal. And a statistically significant correlation was found between genetic mutations of COL4A family numbers and OS (p = 0.0405), DFS (p = 0.0298) in KIRC patients.
Networks Analyses and Functional Enrichment Analysis of COL4A family genes and their Neighboring Genes in KIRC patients.
After confirmed the correlation between genetic mutations in COL4A family numbers and prognosis values, the COL4A's neighbor genes (total 118) obtained from String database was used to construct PPI network to explore the interaction among neighbor genes. And the top ten interacted genes were chosen and highlighted with red by using the plug-in MCODE of Cytoscape (Fig. 8A). As shown in Fig. 8A, the neighbor genes containing COL4A1, COL4A4, COL4A6, COL4A3, COL15A1, HSPG2, COL4A5 and NID1 were the most probably involved in a different expression of COL4A family genes in KIRC patients. Based on 118 neighbor genes, the functional and pathway enrichment analyses were performed to explore the COL4A's biological classification via the online tool Metascape. The COL4A family members and their neighbor genes were significantly involved in collagen − activated tyrosine kinase receptor signaling pathway, collagen − activated signaling pathway, glomerular basement membrane development, retina vasculature development in camera − type eye and glomerulus vasculature development in biologic processes (BP); and collagen type IV trimer, basement membrane collagen trimer, network − forming collagen trimer, collagen network, and complex of collagen trimers in in cellular components (CC); and extracellular matrix structural constituent conferring tensile strength, GABA receptor binding, myosin binding, extracellular matrix structural constituent, and growth factor binding in molecular function (MF); and collagen − activated tyrosine kinase receptor signaling pathway, Anchoring fibril formation, Crosslinking of collagen fibrils, collagen − activated signaling pathway, and glomerular basement membrane development in KEGG pathway.