Bioinformatics Analysis Identifies a Novel Role of GINS1 Gene in Colorectal Cancer

Abstract

Background: Colorectal cancer (CRC) is one of the most lethal malignancies and the incidence of CRC has been on the rise. Herein, we aimed to identify effective biomarkers for early diagnosis and treatment of colorectal cancer via bioinformatic tools.

Methods: To identify differentially expressed genes (DEGs) in CRC, we downloaded CRC gene expression data from GSE24514 and GSE110223 datasets in Gene Expression Omnibus (GEO) and employed R to analyze the data. We further performed functional enrichment analysis of the DEGs on the DAVID gene ontology analysis tool. STRING database and Cytoscape visualization tool were employed to construct a PPI (protein-protein interaction) network and establish intensive intervals in the network. Immunohistochemistry, qRT-PCR and Western blotting were performed to identify the expression level of GINS1 in CRC. In vitro and in vivo experiments were performed to assess the impact of GINS1 in the pathogenesis of CRC in terms of proliferation, migration and metastasis.

Results: Among the two datasets, 389 DEGs were identified and used to construct a PPI network. These genes were mainly involved in cell proliferation and cell cycle. Among them, 15 genes including GINS1 were found to be strongly associated with the PPI network. We further performed immunohistochemistry, qRT-PCR and Western blotting to identify that GINS1 expression was higher in CRC than in paired normal tissues. Moreover, in vitro and in vivo experiments demonstrated GINS1 could promote the proliferation, invasion and migration of colorectal cancer cells.

Conclusions: GINS1 could be considered as a potential biomarker for CRC patients.

Keywords: GINS1; bioinformatics screening; colorectal cancer.

Figure 1

Screen and classify the DEGs in colorectal cancer. (A) DEGs were selected with the threshold of FDR-P<0.01 and |log₂Fold Change|>1 among the GSE24514 dataset. (B) DEGs were selected with the threshold of FDR-P<0.01 and |log₂Fold Change|>1 among the GSE110223 dataset. (C) GSE24514 and GSE110223 datasets showed an overlap of 389 genes. (D) The densest area in the PPI network. (E–H) Functional enrichment analysis including BP, CC, MF and KEGG of the 389 DEGs.

Figure 2

Expression profile and prognostic value of GINS1 in diverse cancers. (A) GINS1 expression in cancers. Expression profile of GINS1 in 33 cancer types compared with adjacent normal tissues by GEPIA. Results revealed that GINS1 was overexpressed in 20 cancer types. T: tumor; N: normal; n: number of patients. (B–I) Upregulation of GINS1 was correlated with poor overall survival and disease-free survival of patients with ACC, KIRP, LIHC, SARC.

Figure 3

Analysis of GINS1 in colorectal cancer. (A) GINS1 was overexpressed in colon adenocarcinoma and rectum adenocarcinoma than in paired normal tissues. *p-value <0.05. (B) Obtain 10 strongly associated co-expressed genes of GINS1 by STRING. (C–D) Genetic alterations of GINS1 and its co-expressed genes in CRC, GINS1 was circled in the picture.

Figure 4

GINS1 was overexpressed in colorectal cancer. (A) Staining of GINS1 in colorectal cancer tissues and paired normal tissues by immunohistochemistry. ***p-value <0.001. (B) Relative expression level of GINS1 in 76 paired human colorectal cancer tissues (T) and the corresponding non-tumor tissues samples (N) are quantified by qRT-PCR. (C) The representative images of protein expression level of GINS1 in colorectal cancer tissues (T) and paired normal tissues (N) by Western blotting. * p-value <0.05. (D) The representative images of protein expression level of GINS1 in different colorectal cancer cell lines and normal colorectal organelle NCM460 cell line by Western blotting. *p-value <0.05, **p-value <0.01.

Figure 5

Overexpression and knockdown of GINS1 in CRC cells. (A) Representative transfected images of SW480 and HCT116 cells. (B and C) Transfection efficiency was validated by qRT-PCR and Western blotting. *p-value <0.05, **p-value <0.01.

Figure 6

In vitro and in vivo experiments demonstrated GINS1 could promote proliferation of CRC cells. (A) Effects of overexpression and knockdown of GINS1 on the formation of colonies in SW480 and HCT116 cell lines. *p-value <0.05, **p-value <0.01. (B) CCK8 results showed that down-regulation of GINS1 reduced the growth of CRC cells, up-regulation of GINS1 promoted the growth of CRC cells. *p-value <0.05, **p-value <0.01. (C) Up-regulation of GINS1 enhanced tumor weight in vivo. **p-value <0.01.

Figure 7

In vitro and in vivo experiments demonstrated GINS1 could promote invasion and migration of CRC cells. (A and B) Transwell test demonstrated knockdown of GINS1 decreased CRC cell invasion and migration abilities, whereas over-expression of GINS1 produced opposite effect. **p-value <0.01, ***p-value <0.001. (C–F) In vivo experiments demonstrated GINS1 promoted metastasis of CRC cells to liver, the metastasis parts are circled in subfigure (C and E).