High expression of collagen 1A2 promotes the proliferation and metastasis of esophageal cancer cells

Abstract

Background: To undertake a bioinformatics analysis to identify abnormally expressed genes [also referred to as differentially expressed genes (DEGs)] and their functions in esophageal carcinoma (ESCA).

Methods: DEGs (i.e., GSE100942, GSE17351, GSE26886, and GSE77861) were obtained from a gene expression omnibus database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using online tools from the Database for Annotation, Visualization and Integrated Discovery. A protein-protein interaction network was then constructed based on the Search Tool for the Retrieval of Interacting Genes website. Cytoscape software was used to identify the top 20 DEGs located in the central region of the network. For the overall survival analysis, a Kaplan-Meier analysis was conducted of the Gene Expression Profiling Interactive Analysis website, and collagen (COL) 1A2 was selected to detect the molecular mechanism of COL1A2-small interfering ribonucleic acid (siRNA) in the following ESCA cell lines: Eca109 and TE-1. Next, the expression of COL1A2-messanger ribonucleic acid was determined using real-time quantitative polymerase chain reaction. The expression of COL1A2 was also verified by Western blot. Cell proliferation was measured by colony-forming and MTT assays, and migration and invasion by the transwell assay.

Results: Based on the GEO database and screening out the hub gene, we identified that COL1A2 was abnormally expressed in ESCA. With a series of in vitro experiments, the expression of COL1A2 was defined as higher in Eca109 and TE-1.

Conclusions: COL1A2 was highly expressed in ESCA tissue samples. Additionally, the proliferation and metastasis of Eca109 and TE-1 cell lines were significantly attenuated by siRNA-COL1A2-mediated small interference. Notably, the expression level of COL1A2 was obviously related to the Akt and epithelial-mesenchymal transition (EMT) pathways.

Keywords: COL1A2; Esophageal carcinoma (ESCA); bioinformatics; extracellular matrix.

Figure 1

Differentially expressed genes (DEGs) identified in GSE100942, GSE17351, GSE26886, and GSE77861. (A) Heatmap of the 414 DEGs according to the value of |logFC|; (B) volcano map of differently expressed genes between esophageal carcinoma (ESCA) tissues and normal esophageal tissues; (C) the top 10 Gene Ontology (GO) terms in the enrichments analysis of the DEGs; (D) the top 10 GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the enrichment analysis of the DEGs.

Figure 2

Differential expression and survival curves of the top 20 hub genes. (A) PPI network of differentially expressed genes (DEGs) based on the Search Tool for the Retrieval of Interacting Genes (STRING) website; (B) the top 20 hub genes were visualized on the Cytoscape application, and the scores (C) were performed; (D) differential expression and survival curves.

Figure 3

Expression of COL1A2 in tissue and cell lines. (A) In four sets of microarray data, GSE100942, GSE17351, GSE26886, and GSE77861, the expression of COL1A2 was higher in cancer tissues than in normal tissues (P<0.05); (B,C) in clinical samples, the mRNA expression of COL1A2 was significantly lower in normal tissues than in tumor tissues; (D) the expression of COL1A2 in cell lines Eca109, TE-1, Ec5, Ec6, and TE-14. *, P<0.05, **P<0.01, ****P<0.0001.

Figure 4

COL1A2 promotes cell colony and growth of Eca109 and TE-1. COL1A2 mRNA (A) was assessed using RT-qPCR after siCOL1A2 small interference (P<0.001). (B,C) Cell colony of Eca109 and TE-1 cells was stained by Crystal violet. (D) Cell growth of Eca109 and TE-1 after COL1A2-siRNA and siRNA-NC infection from day 1 to 5. *P<0.05, ***P<0.001.

Figure 5

Cell invasion of Eca109 and TE-1 cell lines after COL1A2-siRNA and NC-siRNA infection were examined through the transwell assay. Cell migration analysis in Eca109 (A) and TE-1 (B) cells with COL1A2-siRNA and NC-siRNA infection. Cell invasion analysis in Eca109 (C) and TE-1 (D) cells with COL1A2-siRNA and NC-siRNA infection. The samples were stained with crystal violet, and the number of cells was recorded under a high-power microscope (×100). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.