hsa_circ_0000285 sponging miR-582-3p promotes neuroblastoma progression by regulating the Wnt/β-catenin signaling pathway

Abstract

Circular RNA has been reported to play a key role in neuroblastoma (NB); however, the role of circ_0000285 in NB remains unclear. The aim of this study was to elucidate the role of circ_0000285 in NB. We studied the expression patterns of miR-582-3p and circ_0000285 in NB tissues and cells using real-time quantitative polymerase chain reaction. The expression of proteins associated with apoptosis (Bax and Bcl-2) and the proteins associated with Wnt/β-catenin (Wnt, p-Gsk-3β, Gsk-3β, β-catenin, and C-myc) were quantified by western blotting. In vivo animal models were prepared for the functional verification of circ_0000285 on tumor growth. The potential binding of circ_0000285 to miR-582-3p was ascertained using dual-luciferase reporter and RNA-binding protein immunoprecipitation experiments. Noticeable upregulation of circ_0000285 expression was observed in NB tumor samples and cell lines. In vivo and in vitro experiments indicated that the absence of circ_0000285 repressed NB cell proliferation and migration, provoked apoptosis, and impaired the activity of Wnt/β-catenin signaling. miR-582-3p is targeted by circ_0000285 and is poorly expressed in NB cells. The additional repression of miR-582-3p in NB cells after circ_0000285 silencing largely recovered circ_0000285 silencing-suppressed NB cell proliferation and migration and enhanced apoptosis. The absence of miR-582-3p restored Wnt/β-catenin signaling activity impaired by the knockdown of circ_0000285. circ_0000285 functions as an miR-582-3p sponge to strengthen Wnt/β-catenin signaling activity, thus exacerbating NB development.

Keywords: Wnt/β-catenin; circ_0000285; miR-582-3p; neuroblastoma.

Graphical abstract

Figure 1

circ_0000285 showed a high expression level in NB tissues and cells. (a) The relative circ_0000285 expression in the NB and normal samples. (b) The relative circ_0000285 expression in non-cancer control cells (HEK293) and NB cells (SK-N-BE, SK-N-AS, SK-N-SH, and IMR-32), **P < 0.01 vs HEK293. (c) Subcellular distribution of circ_0000285 in nucleus or cytoplasm of SK-N-BE and SK-N-SH cells was ensured by RT-qPCR. (d) The circular structure of circ_0000285 was checked using RNase R, **P < 0.01 vs control.

Figure 2

circ_0000285 silencing restrained NB cell growth in vitro. (a) circ_0000285 expression in si-circ, OE-circ, OE-NC, or si-NC-transfected SK-N-BE and SK-N-SH cells. (b) Cell proliferation in SK-N-SH and SK-N-BE cells after circ_0000285 downregulation or upregulation was evaluated by CCK-8 assay. (c) Cell migration in SK-N-BE and SK-N-SH cells after circ_0000285 absence or presence was detected by wound-healing assay. (d) The protein levels of Bax and Bcl-2 in SK-N-BE and SK-N-SH cells after circ_0000285 absence or presence were ascertained by western blotting. (e) The apoptosis rate in SK-N-BE and SK-N-SH cells after circ_0000285 absence or presence was detected by flow cytometry. **P < 0.01 vs si-NC; ^## P < 0.01 vs OE-NC.

Figure 3

circ_0000285 silencing impaired the activity of Wnt/β-catenin signaling. The levels of wnt, Gsk-3β, β-catenin p-Gsk-3β, and C-myc proteins were measured by Western Blotting. **P < 0.01 vs si-NC; ^## P < 0.01 vs OE-NC.

Figure 4

circ_0000285 silencing repressed tumor development in animal models. (a) The tumor images were taken. The volume (b) and weight of the tumor (c) in the tissue of the tumor in animal models were measured to evaluate the NB tumor growth in vivo. (d) The circ_0000285 expression in the tissues of the tumor in animal models was measured by RT-qPCR. **P < 0.01 vs sh-NC; ^## P < 0.01 vs OE-NC.

Figure 5

circ_0000285 targeted miR-582-3p. (a) The computational binding sites between miR-582-3p and circ_0000285 were acquired from Starbase (https://starbase.sysu.edu.cn/). (b) The putative binding sites between miR-582-3p and circ_0000285 were further verified by dual-luciferase reporter assay, **P < 0.01 vs miR-NC. (c) The binding of circ_0000285 to miR-582-3p was verified by RIP assay, **P < 0.01 vs Anti-IgG. (d) In NB and normal samples, relative miR-582-3p expression was found. (e) The relative miR-582-3p expression in non-cancer cells (HEK293) and NB cells (SK-N-BE and SK-N-SH), **P < 0.01 vs HEK293. (f) The correlation analysis of Spearman examined the association between the expression of miR-582-3p and the expression of circ_0000285 in NB samples.

Figure 6

circ_0000285 knockdown inhibited NB cell functions via promoting miR-582-3p expression. (a–e) Following assays were investigated using SK-N-SH and SK-N-BE cells with si-circ, si-NC, inhibitor, inhibitor-NC or si-circ + inhibitor transfection. (a) The expression of miR-582-3p in these cells was ensured by RT-qPCR. (b) Cell proliferative ability in these cells was checked by CCK-8 assay. (c) Cell migration in these cells was checked by wound-healing assay. (d) In these cells, the expression of Bax and Bcl-2 both proteins was analyzed via Western blotting method. (e) Cell apoptosis in these cells was identified by flow cytometry. *P < 0.05, **P < 0.01 vs si-NC; ^# P < 0.01, ^## P < 0.01 vs inhibitor-NC; ^& P < 0.05, ^&& P < 0.01 vs si-circ + inhibitor.

Figure 7

circ_0000285 knockdown inhibited NB cell functions through Wnt/β-catenin pathway. The protein levels of wnt, p-Gsk-3β, Gsk-3β, β-catenin and C-myc were determined by western blot. *P < 0.05, **P < 0.01 vs si-NC; ^## P < 0.01 vs inhibitor-NC; ^& P < 0.05, ^&& P < 0.01 vs si-circ + inhibitor.