Identification of circ_0000375 and circ_0011536 as novel diagnostic biomarkers of colorectal cancer

Abstract

Background: Colorectal cancer (CRC) imposes a tremendous burden on human health, with high morbidity and mortality. Circular ribonucleic acids (circRNAs), a new type of noncoding RNA, are considered to participate in cancer pathogenesis as microRNA (miRNA) sponges. However, the dysregulation and biological functions of circRNAs in CRC remain to be explored.

Aim: To identify potential circRNA biomarkers of CRC and explore their functions in CRC carcinogenesis.

Methods: CircRNAs and miRNAs differentially expressed in CRC tissues were identified by analyzing expression profiles from the Gene Expression Omnibus (GEO) database. Circ_0000375 and circ_0011536 were selected as CRC biomarker candidates. Quantitative real-time polymerase chain reaction was utilized to evaluate the expression of these 2 circRNAs in CRC tissues, serums and cell lines. Receiver operating characteristic curves were generated to assess the diagnostic performances of these 2 circRNAs. Then, functional experiments, including cell counting kit-8, wound healing and Transwell invasion assays, were performed after the overexpression of circ_0000375 and circ_0011536 in CRC cell lines. Furthermore, candidate target miRNAs of circ_0000375 and circ_0011536 were predicted via bioinformatics analysis. The expression levels of these miRNAs were explored in CRC cell lines and tissues from GEO datasets. A luciferase reporter assay was developed to examine the interactions between circRNAs and miRNAs. Based on the target miRNAs and downstream genes, functional enrichment analyses were applied to reveal the critical signaling pathways involved in CRC carcinogenesis.

Results: Downregulated circ_0000375 and circ_0011536 expression was observed in CRC tissues in GSE126095, clinical CRC tissue and serum samples and CRC cell lines. The areas under the curve for circ_0000375 and circ_0011536 were 0.911 and 0.885 in CRC tissue and 0.976 and 0.982 in CRC serum, respectively. Moreover, the serum levels of these 2 circRNAs were higher in patients at 30 d postsurgery than in patients before surgery, suggesting that the serum expression of circ_0000375 and circ_0011536 is related to CRC tumorigenesis. Circ_0000375 and circ_0011536 overexpression inhibited the proliferation, migration and invasion of CRC cells. Furthermore, miR-1182 and miR-1246, which were overexpressed in CRC tissues in GSE41655, GSE49246 and GSE115513, were verified as target miRNAs of circ_0000375 and circ_0011536, respectively, by luciferase reporter assays. The downstream genes of miR-1182 and miR-1246 were enriched in some CRC-associated pathways, such as the Wnt signaling pathway.

Conclusion: Circ_0000375 and circ_0011536 may function as tumor suppressors in CRC progression, serving as novel biomarkers for CRC diagnosis and as promising candidates for therapeutic exploration.

Keywords: Biomarker; Cir_0000375; Circ_0011536; Colorectal cancer; MicroRNA; Tumor suppressor.

Figure 1

Expression profiles of circular ribonucleic acids in colorectal cancer tissues from GSE126095 datasets and in cell lines. A: Volcano plot of circular ribonucleic acids expression levels; B: The expression of circ_0000375 in colorectal cancer (CRC) tissues and negative control tissues; C: The expression of circ_0011536 in CRC tissues and negative control tissues; D: Receiver operating characteristic curves to evaluate the diagnostic value of circ_0000375 and circ_0011536 by assessing the area under the curve; E: The relative expression levels of circ_0000375 in a normal colon cell line (FHC) and CRC cell lines (HCT116, RKO and SW480); F: The relative expression levels of circ_0011536 in a normal colon cell line (FHC) and CRC cell lines (HCT116, RKO and SW480). ^aP < 0.001; ^bP < 0.01; ^cP < 0.05. NC: Negative control; AUC: Area under the curve; CRC: Colorectal cancer.

Figure 2

Characteristics of circ_0000375 and circ_0011536 in colorectal cancer tissues. A: Structures and back-splicing sequences of circ_0000375; B: Structures and back-splicing sequences of circ_0011536; C: The relative expression levels of circ_0000375 and the corresponding linear mRNA IFFO1 in colorectal cancer tissues treated with RNase R; D: The relative expression levels of circ_0011536 and ZMYM4 treated with RNase R. ^bP < 0.01; ^cP < 0.05. NS: Not significant.

Figure 3

Circ_0000375 and circ_0011536 were downregulated in colorectal cancer tissue and serum samples. A: The expression levels of circ_0000375 in healthy control (HC) and colorectal cancer (CRC) tissue samples; B: The expression levels of circ_0011536 in HC and CRC tissue samples; C: Receiver operating characteristic curves for circ_0000375, circ_0011536 and combined circular ribonucleic acids in CRC tissues with the area under the curve calculated; D: The expression levels of circ_0000375 in serum samples of HC, preoperative CRC and CRC after surgery; E: The expression levels of circ_0011536 in serum samples of HC, preoperative CRC and CRC after surgery; F: Receiver operating characteristic curves for Circular ribonucleic acids in CRC serum samples. ^aP < 0.001; ^bP < 0.01; ^cP < 0.05. CRC: Colorectal cancer; HC: Healthy control; AUC: Area under the curve; ROC: Receiver operating characteristic.

Figure 4

Overexpression of circ_0000375 and circ_0011536 suppresses proliferation, migration and invasion in colorectal cancer. A: The relative expression levels of circ_0000375 and circ_0011536 in SW480 cells transfected with overexpression plasmids; B: The relative expression levels of circ_0000375 and circ_0011536 in HCT116 cells transfected with overexpression plasmids; C: Cell counting kit-8 (CCK-8) assays were performed to determine the ability of proliferation in SW480 cells transfected with oe-circ_0000375 or oe-circ_0011536; D: CCK-8 assays were performed to determine the ability of proliferation in HCT116 cells transfected with oe-circ_0000375 or oe-circ_0011536; E: Wound healing assays were conducted to assess the cell migrative capability of SW480; F: Wound healing assays were conducted to assess the cell migrative capability of HCT116 cells; G: Transwell invasion assays were utilized to reveal the invasive ability of SW480; H: Transwell invasion assays were utilized to reveal the invasive ability of HCT116 cells; I: Results of wound healing assays of SW480 and HCT116 cell lines; J: Results of Transwell invasion assays of SW480 and HCT116 cell lines. ^aP < 0.001; ^bP < 0.01; ^cP < 0.05.

Figure 5

Circ_0000375 and circ_0011536 serve as sponges of miR-1182 and miR-1246 in colorectal cancer. A: The expression level of miR-1182 in a normal colon cell line (FHC) and colorectal cancer cell lines (HCT116, RKO and SW480); B: The relative expression level of miR-1182 in SW480 and HCT116 cells transfected with a circ_0000375 overexpression vector; C: Relative luciferase activities were detected in HEK293T cells after cotransfection with circ_0000375-WT, circ_0000375-MUT, miR-1182 mimics and negative control mimics; D: The expression level of miR-1246 in colorectal cancer cell lines; E: The relative expression level of miR-1246 in SW480 and HCT116 cells transfected with oe-circ_0011536; F: Luciferase reporter assays were performed after cotransfection with circ_0011536-WT, circ_0011536-MUT, miR-1246 mimics and negative control mimics. ^aP < 0.001; ^bP < 0.01; ^cP < 0.05. NC: Negative control; NS: Not significant.

Figure 6

MiR-1182 and miR-1246 were upregulated in colorectal cancer tissues in Gene Expression Omnibus datasets. A: The expression level of miR-1182 in GSE41655; B: The expression level of miR-1182 in GSE49246; C: The expression level of miR-1182 in GSE115513; D: The expression level of miR-1246 in GSE41655; E: The expression level of miR-1246 in GSE49246; F: The expression level of miR-1246 in GSE115513; G: The diagnostic value of miR-1182 in Gene Expression Omnibus (GEO) datasets was evaluated by the area under the curve; H: The diagnostic value of miR-1246 in GEO datasets was evaluated by the area under the curve. ^aP < 0.001; ^bP < 0.01; ^cP < 0.05. CRC: Colorectal cancer; NC: Negative control; AUC: Area under the curve.

Figure 7

Enrichment analyses based on downstream genes of miR-1182 and miR-1246. A: Gene Ontology analysis of biological processes, cellular components, and molecular functions; B: Kyoto Encyclopedia of Genes and Genomes pathway analysis.