Expression signature and prognostic value of CREC gene family in human colorectal cancer

Abstract

Colorectal cancer (CRC) is one of the malignant tumors with the highest morbidity and mortality and poor prognosis. The mammalian gene family of Cab45/reticulocalbin/ERC-45/calumenin (CREC) consists of RCN1, RCN2, RCN3, SDF4 and CALU. Although CREC family members have been associated with CRC, the expression pattern, prognostic value, and the role of CREC family in CRC remain unclear. In this study, the expression, survival and biological functions of CREC family in CRC were determined via bioinformatic datasets analysis and experimental verification on clinical CRC specimen. Bioinformatic analysis showed that the expression levels of most CREC family genes were higher in CRC tissues than in normal colorectal tissues. The qPCR and western blot results also revealed that the transcriptional and protein levels of CREC family were elevated in CRC tissues compared with adjacent tissues. Besides, CREC family was significantly correlated with advanced tumor stage and poor prognosis of CRC patients. The expression levels of CREC family had correlations with genomic mutation and methylation, and with the infiltration levels of CD4 + T cells, macrophages, neutrophils, and dendritic cells in the microenvironment of CRC. Functional networks enrichment analysis indicated that the genes of CREC family were essential factors for CRC metastasis. Collectively, these findings suggest that CREC family might be potential targets for the treatment of CRC and candidate prognostic markers for CRC patients.

Keywords: Bioinformatics; CREC family; Colorectal cancer; Expression signature; Prognosis.

Fig. 1

The mRNA expression patterns of RCN1, RCN2, RCN3, SDF4 and CALU in different types of human cancer. A Analysis results based on the Oncomine database. B Analysis results based on the GEPIA database

Fig. 2

Expression level of CREC family in CRC tissues. A Analysis results based on the GEPIA database. B Analysis results based on the UALCAN Database

Fig. 3

Representative immunohistochemical stains of distinct CREC family members in CRC tissues and adjacent normal tissues (derived from Human Protein Atlas)

Fig. 4

Elevated expressions of CREC gene family in CRC cell lines and tissues. A The mRNA levels of CREC family in normal colonic epithelial cell NCM460 and CRC cell lines (SW480, SW620, and HCT116) detected by qPCR (n = 3). B The mRNA levels of CREC family in CRC tissues (T1-T5) and normal adjacent tissues (N1-N5) from 11 CRC patients. C The protein levels of CREC family in CRC tissues and normal adjacent tissues from 11 CRC patients. Data were presented as the mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 5

TFs-target and miRNAs-target that regulate CREC family. A TFs-target result (derived from hTFtarget). B miRNAs-target result of the intersection selected from Starbase and Targetscan databases (C) Detailed miRNAs-target result (derived from Starbase and Targetscan)

Fig. 6

Significance of CREC expression in tumor stage and prognosis of CRC. A Correlation between the mRNA levels of CREC family and tumor stage in CRC patients (derived from UALCAN). B, C The prognostic value of mRNA levels of CREC family in CRC patients. Data were derived from Kaplan–Meier Plotter and GEPIA database

Fig. 7

ROC curve of the risk score in inferior prognosis prediction of CREC family genes for 5-year overall survival in CRC

Fig. 8

Knocking down RCN1 in SW480 cells effectively inhibited cell migration. A, B After knocking down RCN1 in SW480 cells, the scratch healing rate was measured. C-E Western blot analysis of E-Cadherin and Vimentin in SW480 cells with knockdown of RCN1. * p < 0.05, ** p < 0.01, n = 3

Fig. 9

Correlation of CREC family expression with immune cell infiltration levels in CRC. A Analysis result derived from Timer. B Immunohistochemical images of CD206 from clinical CRC tissues and adjacent tissues included in this study. Scale bar: 100 μm. Data were presented as the mean ± SEM, ** p < 0.01

Fig. 10

Analysis of CREC family gene mutation, expression and DNA methylation in CRC. A CREC family gene mutation analysis in CRC (derived from cBioPortal). B Correlation between gene expressions of CREC family and DNA methylation in CRC (derived from cBioPortal)

Fig. 11

Relationships among CREC family and co-expressed genes of CREC family in CRC. A Potential correlations among RCN1, RCN2, RCN3, SDF4 and CALU. B The network for CREC family and the top 50 co-expression genes

Fig. 12

The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on the functions of CREC family and the top 50 co-expression genes