Circ_0082182 promotes oncogenesis and metastasis of colorectal cancer in vitro and in vivo by sponging miR-411 and miR-1205 to activate the Wnt/β-catenin pathway

Abstract

Background: Circular RNAs (circRNAs) are a class of endogenous single-strand RNA transcripts with crucial regulation in human cancers. The objective of this study is to investigate the role of circ_0082182 in CRC and its specific functional mechanism.

Methods: The quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the levels of circ_0082182, microRNA-411 (miR-411) and microRNA-1205 (miR-1205). Cell proliferation was detected by Cell counting Kit-8 (CCK-8) and colony formation assays. Flow cytometry was used for determining cell cycle and cell apoptosis. Cell apoptosis was also assessed by caspase3 and caspase9 activities. Cell migration and invasion were examined using scratch assay and transwell assay. The interaction between circ_0082182 and miRNA was validated by the dual-luciferase reporter and biotinylated RNA pull-down assays. Wnt/β-catenin pathway and epithelial-mesenchymal transition (EMT)-associated proteins were quantified by Western blot. Xenograft model was established for the research of circ_0082182 in vivo.

Results: Circ_0082182 was upregulated in CRC and could predict the poor prognosis of CRC patients. Functionally, circ_0082182 promoted CRC cell proliferation, cell cycle progression, and metastasis while inhibited apoptosis. Subsequently, circ_0082182 was shown to act as the sponges of miR-411 and miR-1205. MiR-411 and miR-1205 were identified as tumor inhibitors in CRC. Furthermore, circ_0082182 promoted the CRC progression via sponging miR-411 and miR-1205. Moreover, circ_0082182 facilitated the Wnt/β-catenin pathway and EMT process by targeting miR-411 and miR-1205. In vivo, circ_0082182 accelerated the CRC tumorigenesis and EMT process by activating the Wnt/β-catenin pathway by downregulating the expression of miR-411 or miR-1205.

Conclusion: This study showed that circ_0082182 functioned as an oncogene in the developing process of CRC by sponging miR-411 or miR-1205 to activate the Wnt/β-catenin pathway. Circ_0082182 might be a molecular target in the diagnosis and treatment of CRC.

Keywords: Colorectal cancer; Wnt/β-catenin pathway; circ_0082182; miR-1205; miR-411.

Fig. 1

Circ_0082182 was upregulated in CRC and associated with a poor prognosis. a Circ_0082182 was upregulated in CRC tissues compared with normal tissues by qRT-PCR analysis. b High circ_0082182 expression could predict the poor survival of CRC patients. c Circ_0082182 level was higher in CRC (HCT116, SW480, SW620 and CaCo-2) cells than that in normal NCM-460 cells. **P < 0.01

Fig. 2

Circ_0082182 contributed to cell proliferation and cell cycle progression while reduced apoptosis in CRC cells. a Circ_0082182 expression was knocked down by sh-circ in HCT116 and CaCo-2 cells. b–d Cell proliferation by CCK-8 assay (b, c) and colony formation (d) assay was inhibited after circ_0082182 was downregulated. e, f Cell cycle progression by flow cytometry was blocked by the knockdown of circ_0082182. g, i Cell apoptotic rate (g, h) and caspase3/caspase9 activities (i) were increased in sh-circ group relative to sh-NC group. *P < 0.05, **P < 0.01.

Fig. 3

Circ_0082182 functioned as a metastasis-promoting factor in CRC cells. a, b Downregulation of circ_0082182 reduced the cell migratory rate in the scratch assay (a) and the invaded cell number in the transwell assay (b). **P < 0.01

Fig. 4

Circ_0082182 targeted miR-411 and miR-1205 in CRC cells. a, b Circinteractome showed the binding sites between circ_0082182 and miR-411 (a) or miR-1205 (b). c, e The interaction between circ_0082182 and miR-411 or miR-1205 was validated using the dual-luciferase reporter assay (c, d) and Bio-coupled RNA pull-down assay (e). f, g The expression levels of miR-411 and miR-1205 were downregulated in CRC cells (f) and tissues (g). h Knockdown of circ_0082182 induced the upregulation of miR-411 and miR-1205 in HCT116 and CaCo-2 cells. **P < 0.01

Fig. 5

MiR-411 and miR-1205 retarded the progression of CRC. a MiR-411 and miR-1205 levels were overexpressed by transfection of miR-411 and miR-1205. b–d Cell proliferation (b, c) and cell cycle (d) were suppressed in miR-411 or miR-1205 group contrasted with miR-NC group. e, f Transfection of miR-411 or miR-1205 enhanced the apoptotic rate (e) and the caspase3/caspase9 activities (f). g, h Overexpression of miR-411 or miR-1205 triggered the inhibition of cell migration (g) and invasion (h). **P < 0.01

Fig. 6

Circ_0082182 acted as a carcinogene in CRC by sponging miR-411 and miR-1205. a The inhibitory efficiencies of anti-miR-411 and anti-miR-1205 were shown to be great via qRT-PCR detection. b–d Inhibitor of miR-411 or miR-1205 eliminated the sh-circ-induced proliferation inhibition (b, c) and cell cycle arrest (d). e, f The promoting effects of sh-circ on cell apoptotic rate (e) and caspase3/caspase9 activities (f) were relieved by anti-miR-411 or anti-miR-1205. g, h Sh-circ-mediated inhibition of cell migration (g) and invasion (h) was restored by miR-411 or miR-1205 inhibitor. *P < 0.05, **P < 0.01

Fig. 7

Circ_0082182 sponged miR-411 and miR-1205 to activate Wnt/β-catenin pathway and promote EMT process in CRC cells. a, b Knockdown of circ_0082182 repressed the Wnt/β-catenin pathway and EMT process in HCT116 and CaCo-2 cells by upregulating the level of miR-411 or miR-1205. **P < 0.01

Fig. 8

Circ_0082182 facilitated CRC tumorigenesis and EMT in vivo by activating the miR-411/miR-1205-mediated Wnt/β-catenin pathway. a Tumor volume was lower in sh-circ group than that in sh-NC group. b Tumors were excised and tumor weight was inhibited in sh-circ group by comparison with sh-NC group. c, d The circ_0082182 expression was downregulated (c) and miR-411/miR-1205 levels were increased (d) by sh-circ. e Silence of circ_0082182 inactivated the Wnt/β-catenin pathway and EMT process in tumor tissues. **P < 0.01