Circ_0005927 Inhibits the Progression of Colorectal Cancer by Regulating miR-942-5p/BATF2 Axis

Abstract

Background: Colorectal cancer (CRC) is one of the most common aggressive neoplasms worldwide. Circular RNAs (circRNAs) have been involved in the biological process of CRC. This study aimed to explore the effects of circ_0005927 on CRC progression and underneath mechanism.

Materials and methods: The expression of circ_0005927, microRNA-942-5p (miR-942-5p) and basic leucine zipper ATF-like transcription factor 2 (BATF2) was detected by quantitative real time polymerase chain reaction (qRT-PCR). The protein expression of BATF2 was determined by Western blot. The effects among circ_0005927, miR-942-5p and BATF2 on cell colony-forming ability, apoptosis and migratory and invasive abilities were revealed by cell colony formation, flow apoptosis and transwell assays, respectively. The associated relationship between miR-942-5p and circ_0005927 or BATF2 was predicted by Circinteractome or TargetScan online database, and identified by dual-luciferase reporter or RNA immunoprecipitation (RIP) assay. The impacts of circ_0005927 overexpression on tumor growth in vivo were investigated by in vivo tumor formation assay.

Results: Circ_0005927 expression and the mRNA and protein expression of BATF2 were dramatically downregulated, while miR-942-5p expression was obviously upregulated in CRC tissues or cells compared with control groups. Circ_0005927 overexpression repressed cell colony-forming ability, migration and invasion, whereas induced cell apoptosis in CRC; however, these impacts were hindered by miR-942-5p mimic or BATF2 knockdown. Furthermore, circ_0005927 was a sponge of miR-942-5p, and miR-942-5p bound to BATF2. In addition, circ_0005927 overexpression repressed tumor growth in vivo.

Conclusion: Circ_0005927 suppressed cell colony-forming ability, migration and invasion, and promoted cell apoptosis by sponging miR-942-5p to induce BATF2 in CRC. The possible mechanism provides a theoretical basis for the study of circRNA-directed therapy for CRC.

Keywords: BATF2; CRC; circ_0005927; circular RNA; miR-942-5p.

Figure 1

Circ_0005927 expression was dramatically downregulated in CRC tissues and cells and was correlated with poor clinical outcome. (A and B) GSE142837 microarray dataset and qRT-PCR showed that circ_0005927 expression was lower in CRC tissues than that in healthy intestinal tissues. (C and D) Circ_0005927 expression was detected by qRT-PCR in CRC tissues, normal intestinal tissues, NCM460 cells, SW620 cells, SW480 cells, Caco-2 cells and LoVo cells. (E and F) RNase R treatment assay was employed to demonstrate that circ_0005927 was more stable than linear GAPDH. *P<0.05 and **P<0.01.

Figure 2

Circ_0005927 suppressed cell colony-forming ability and promoted cell apoptosis in CRC. (A and B) The efficiency of Lv-circ_0005927 and si-circ_0005927 in affecting circ_0005927 expression was detected by qRT-PCR in LoVo and SW620 cells. (C and D) Cell colony forming assay was performed to explain the effects of circ_0005927 overexpression and knockdown on cell colony-forming ability in LoVo and SW620 cells. (E and F) The impacts of circ_0005927 overexpression and silencing on the apoptosis of LoVo and SW620 cells were explored by flow cytometry analysis. **P<0.01.

Figure 3

Circ_0005927 restrained the migration and invasion of CRC cells. (A and B) Transwell assay was conducted to determine the influences of Lv-circ_0005927 and si-circ_0005927 on cell migratory and invasive abilities in LoVo and SW620 cells. **P<0.01.

Figure 4

Circ_0005927 bound to miR-942-5p in LoVo and SW620 cells. (A) The binding sequence between circ_0005927 and miR-942-5p was predicted by Circinteractome online database. (B) MiR-942-5p expression was detected by qRT-PCR after miR-942-5p mimic transfection. (C and D) Dual-luciferase reporter assay was employed to detect luciferase activities in LoVo and SW620 cells. (E and F) RIP assay was performed to confirm that circ_0005927 was associated with miR-942-5p in LoVo and SW620 cells. (G and H) The expression level of miR-942-5p was determined by qRT-PCR in NCM460 cells, LoVo cells, SW620 cells and CRC tissues and normal intestinal tissues. (I) The linear relationship between circ_0005927 expression and miR-942-5p expression was revealed by Spearman correlation analysis. **P<0.01.

Figure 5

Circ_0005927 sponged miR-942-5p to regulate BATF2. (A) The binding sites between miR-942-5p and BATF2 were predicted by TargetScan online database. (B and C) Dual-luciferase reporter assay was performed to verify that miR-942-5p bound to BATF2. (D) Western blot was carried out to detect the protein expression of BATF2 in NCM460, LoVo and SW620 cells. (E) QRT-PCR was employed to determine BATF2 mRNA expression in CRC tissues and normal intestinal tissues. (F and G) The linear relationship between BATF2 expression and circ_0005927 expression or miR-942-5p expression was explained by Spearman correlation analysis. (H) The interfering efficiency of anti-miR-942-5p was determined by qRT-PCR. (I) The effect of miR-942-5p inhibitor on the protein expression of BATF2 was disclosed by Western blot. (J) The impacts between circ_0005927 overexpression and miR-942-5p mimic on BATF2 protein expression were unveiled by Western blot. **P<0.01.

Figure 6

Circ_0005927 overexpression repressed CRC cell processes by regulating miR-942-5p and BATF2. (A) Western blot was used to determine the knockdown efficiency of si-BATF2 in LoVo and SW620 cells. (B) The effects between circ_0005927 and miR-942-5p mimic or BATF2 silencing on cell colony-forming ability were revealed by cell colony formation assay in LoVo and SW620 cells. (C) Flow cytometry analysis was performed to investigate the impacts between circ_0005927 overexpression and miR-942-5p mimic or BATF2 depletion on cell apoptosis in LoVo and SW620 cells. (D and E) Transwell assay was employed to display the influences between circ_0005927 and miR-942-5p mimic or BATF2 absence on the migration and invasion of LoVo and SW620 cells. **P<0.01.

Figure 7

Circ_0005927 overexpression repressed tumor growth in vivo. (A and B) The effects of circ_0005927 overexpression on tumor volume and weight were revealed in vivo. (C) QRT-PCR was performed to determine the impacts of circ_0005927 overexpression on the expression of circ_0005927 and miR-942-5p. (D) Western blot was employed to unveil the influence of circ_0005927 overexpression on BATF2 protein expression. **P<0.01.