The analyses of SRCR genes based on protein-protein interaction network in esophageal squamous cell carcinoma

Abstract

The scavenger receptor cysteine-rich (SRCR) proteins, with one to several SRCR domains, play important roles in human diseases. A full view of their functions in esophageal squamous cell carcinoma (ESCC) remain unclear. Sequence alignment and phylogenetic tree for all human SRCR domains were performed. Differentially-expressed SRCR genes were identified in ESCC, followed by protein-protein interaction (PPI) network construction, topological parameters, subcellular distribution, functional enrichment and survival analyses. The variation of conserved cysteines in each SRCR domain suggested a requirement for new classification of the SRCR family. Six genes (LGALS3BP, MSR1, CD163, LOXL2, LOXL3 and LOXL4) were upregulated, and four genes (DMBT1, PRSS12, TMPRSS2 and SCARA5) were downregulated in ESCC. These 10 SRCR genes form a unique biological network. Functional enrichment analyses provided important clues to investigate the biological functions for SRCR gene network in ESCC, such as extracellular structure organization and the PI3K-Akt signaling pathway. Kaplan-Meier curves confirmed that high expression of SCARA5, LOXL2, LOXL3, LOXL4 were related to poor survival, whereas high expression of DMBTI and PRSS12 showed the opposite result. SRCR genes promote the development of ESCC through its network and could serve as potential prognostic factors and therapy targets of ESCC.

Keywords: SRCR gene; esophageal squamous cell carcinoma; functional enrichment; protein-protein interaction network; sequence analysis.

Figure 1

Sequence analyses of SRCR genes. A. Protein sequence alignment of a total of 91 human SRCR domains, which were extracted from 27 human SRCR proteins and considered as a single unit. Identical and amino acid residues with similar physical and chemical properties are indicated by the same color. The eight conserved cysteines are indicated in red blocks. B. Cluster based on sequence similarities. These SRCR domains are approximately classified into four groups, indicating a gradual variation. C. The phylogenetic tree based on the protein sequences of SRCR domains.

Figure 2

Heatmap of differentially-expressed SRCR genes in three microarray datasets. A-C. Hierarchical clustering for differentially-expressed SRCR genes based on their expression levels in GSE23400, GSE53622 and GSE53624, respectively. A gradient of red to yellow color indicates high to low expression. Red bars and green bars in the left panel represent ESCC clinical samples and matched normal tissues, respectively. Each row represents a clinical sample.

Figure 3

Three PPI networks for differentially-expressed SRCR genes. A. The “Full SRCR-PPIN” showing the interactions between each protein pair in the network. SRCR proteins are indicated by a deep blue color, and their interacting proteins are shown in light blue. B. “Concise SRCR-PPIN” indicates many SRCR genes are linked through one interacting protein, indicated by the light pink color. C. The log2 (fold change) and co-expression correlation coefficient for proteins integrated into the Concise SRCR-PPIN. The red or green node indicates their upregulation and downregulation in expression. Non-significantly changed proteins remain as blue. The red or green line between nodes indicates a positive or negative correlation, respectively. Thicker lines indicate higher correlation coefficients.

Figure 4

Topological properties of the Full SRCR-PPIN. A. The node degree distribution of the Full SRCR-PPIN, indicating a power-law distribution character. B. Shortest path length of the Full SRCR-PPIN. The frequency suggests the number of protein pairs have a certain shortest path length. C. Neighbor connectivity of the Full SRCR-PPIN. The decreasing distribution indicates the edges between sparsely connected and highly connected nodes prevail in the network. D. Closeness centrality distribution of the Full SRCR-PPIN. The closeness centrality of each node varies between 0 and 1.

Figure 5

Subcellular localization distribution of the Full SRCR-PPIN. Differentially-expressed SRCR genes are indicated by a black box. The Concise-SRCR network was spread into layers according to location of proteins, which looks like non-canonical signal pathways.

Figure 6

Functional enrichment analyses of the Full SRCR-PPIN. A. A functional-grouped network was generated from Gene Ontology (GO) “Biological Process” enrichment analysis. The 67 GO terms were linked when their overlapped kappa score was ≥ 0.3. Similar GO terms are labeled in the same or similar color. B. KEGG pathways involving proteins in the network were analyzed by the hyper-geometric distribution. Node sizes represent the term enrichment significance.

Figure 7

The expression level of differentially-expressed SRCR genes correlates with survival time of ESCC patients. The left panel of each gene shows its relative expression level after optimized classification by the X-tile program. The right panel of each gene indicates the amount of classification, as well as the hazard ratio.

Figure 8

SRCR domains are important for the integrity of LOXL2 function. A. The significant GO term “collagen fibril organization”generated from LOXL2 overexpression, which contains 6 gene with their absolute fold-change more than 1. B. Schematic presentation of LOXL2 functional domains and their protein expression. C, D. Comparison of the ability to promote migration and invasion for full-length and truncated LOXL2, respectively. The abilities of migration and invasion of cells caused by truncated LOXL2 were much weaker than that with full length LOXL2. E. Colony formation ability of ESCC cells expressing truncated LOXL2 was less than full length LOXL2 in ESCC cells.