CircRNA_002178 as a ceRNA promotes the development of colorectal cancer by regulating miR-542-3p/CREB1

Abstract

Objective: Colorectal cancer (CRC) is a malignant tumor commonly found in the digestive tract. This study aimed to explore the effect of circRNA_002178 as a competing endogenous RNA in the development of CRC by regulating the miR-542-3p/cAMP response element binding protein 1 (CREB1) axis and its molecular mechanism.

Methods: The relative expressions of circ_002178, miR-542-3p, and CREB1 in patients' cell lines and CRC tissues were measured using Western blot and qRT-PCR. The localization and expression of circ_002178 were determined using fluorescence in situ hybridization and nucleocytoplasmic separation tests. The targeting relationships among circ_002178, miR-542-3p, and CREB1 were validated using RNA immunoprecipitation and dual luciferase reporter assays. The cells' proliferation, invasion, and colony forming ability were tested using CCK8, Transwell, and Clone formation assays, respectively. The cellular glucose consumption, lactification, and adenosine triphosphate (ATP) production were measured using glucose uptake colorimetric assay kits, lactate colorimetric assay kits and ATP assay kits, respectively.

Results: The circ_002178 and CREB1 expressions were up-regulated in the CRC cells and tissues, and the miR-542-3p expression was down-regulated (all P<0.05). The circ_002178 knockdown inhibited the proliferation, invasion, colony formation, and glycolysis of the CRC cells in vitro, but the overexpression of circ_002178 induced the opposite result (both P<0.05). Our molecular mechanism study revealed that circ_002178, as the molecular sponge of miR-542-3p, promotes CREB1 expression. The downregulation of miR-542-3p or the overexpression of CREB1 is able to partly weaken the inhibition of CRC cells through the circ_002178 knockdown.

Conclusion: circ_002178 promotes the invasion, proliferation, colony formation, and glycolysis of CRC cells by regulating the miR-542-3p/CREB1 axis, thus driving the development of CRC.

Keywords: Colorectal cancer; cAMP response element binding protein 1; circ_002178; glycolysis; miR-542-3p.

Figure 1

circ_002178 expression profile. A: The GSE126095 data set analysis results (Normal: N=10, Cancer: N=10); B: The circ_002178 expression in tissues (Normal: N=60, CRC: N=60); C: circ_002178 sensitivity analyzed by the ROC curve. Compared with the normal group, ^aP<0.05. CRC: colorectal cancer; ROC: receiver operating characteristics; AUC: area under the curve.

Figure 2

circ_002178 was highly expressed in the CRC cells and participated in the CRC cell proliferation and colony formation. A: The circ_002178 expressions in cells; B: The plasmid transfection efficiency measured using qRT-PCR; C: The cell proliferation ability; D: The cell colony forming ability. Compared with the NCM460 group, ^bP<0.05; compared with the NC group, ^cP<0.05. NC: negative control; OD: optical density; CRC: colorectal cancer; qRT-PCR: quantitative real-time polymerase chain reaction.

Figure 3

The participation of circ_002178 in the CRC cell invasion and glycolysis. A: The cell invasion ability (200×); B: The glucose consumption level; C: The lactate production; D: The ATP production. Compared with the NC group, ^cP<0.05. NC: negative control; CRC: colorectal cancer; ATP: adenosine triphosphate.

Figure 4

circ_002178 functions as a molecular sponge for miR-542-3p. A: The FISH results (400×); B: The expression of circ_002178 in the cytoplasms and nuclei; C: A stability test for circ_002178; D: circ_002178 and its target miRNAs; E: KEGG analysis results of the target miRNA for circ_002178; F, G: The expression of miR-542-3p in the tissues and cells (in tissues, Normal: N=60, CRC: N=60); H: A correlation analysis of circ_002178 and miR-542-3p (N=60); I: The effect of circ_002178 on the miR-542-3p expression; J: The specific binding sites of circ_002178 and miR-542-3p; K, L: The targeting relationship between circ_002178 and miR-542-3p validated using dual luciferase reporter and RIP assays. Compared with the Nucleus group, ^dP<0.05; compared with the RNase R (-) group, ^%P<0.05; compared with the Normal group, ^aP<0.05; compared with the NCM460 group, ^bP<0.05; compared with the NC group, ^cP<0.05; compared with the miR-NC+WT group, ^&P<0.05; compared with the IgG group, ^@P<0.05. NC: negative control; CRC: colorectal cancer; FISH: fluorescence in situ hybridization; KEGG: Kyoto Encyclopedia of Genes and Genomes; RIP: RNA immunoprecipitation assay.

Figure 5

The targeting relationship between miR-542-3p and CREB1. A: The intersection of the StarBase and miRWalk online websites in predicting the target genes; B: The KEGG pathway enrichment analysis; C: The protein-protein interaction analysis; D: The specific binding sites of miR-542-3p and CREB1; E, F: The targeting relationship between miR-542-3p and CREB1 validated using a dual luciferase reporter assay and an RIP assay. Compared with the miR-NC+WT group, ^&P<0.05; compared with the IgG group, ^@P<0.05. NC: negative control; KEGG: Kyoto Encyclopedia of Genes and Genomes; RIP: RNA immunoprecipitation assay.

Figure 6

The regulatory effect of circ_002178/miR-542-3p on CREB1. A, B: The expression of CREB1 in the tissues and cells (in tissues, Normal: N=60, CRC: N=60); C, D: Effect of circ_002178/miR-542-3p on CREB1 expression; E, F: Correlation analysis between circ_002178/miR-542-3p and CREB1 (N=60). Compared with the Normal group, ^aP<0.05; compared with the NCM460 group, ^bP<0.05; compared with the NC group, ^cP<0.05; compared with the miR-NC group, *P<0.05. NC: negative control; CRC: colorectal cancer.

Figure 7

The knockdown of circ_002178 inhibited the CRC cell proliferation and clone formation by the miR-542-3p/CREB1 axis. A: The cell proliferation ability; B: The cell colony forming ability. Compared with the NC group, ^cP<0.05; compared with the si-circ_002178+miR-542-3p inhibitor group, ^P<0.05. NC: negative control; CRC: colorectal cancer.

Figure 8

The knockdown of circ_002178 inhibited the CRC cell invasion and glycolysis by the miR-542-3p/CREB1 axis. A: The cell invasion ability (200×); B: The glucose consumption; C: The lactate production; D: The ATP production. Compared with the NC group, ^cP<0.05; compared with the si-circ_002178+miR-542-3p inhibitor group, ^P<0.05. NC: negative control; CRC: colorectal cancer; ATP: adenosine triphosphate.

Figure 9

circRNA_002178, as a ceRNA, affects the proliferation, invasion, cloning, and aerobic glycolysis of colorectal cancer cells by regulating the miR-542-3p/CREB1 axis. ceRNA: competing endogenous RNA.