Circular RNA hsa_circ_0000658 inhibits osteosarcoma cell proliferation and migration via the miR-1227/IRF2 axis

Abstract

Osteosarcoma (OS) is the most frequently occurring bone cancer. Circular RNAs (circRNAs) have been shown to exert pivotal impact in modulation of gene expression, but their roles in OS are still not fully understood. In this study, we analysed the role of circ-0000658 in OS. Thereafter, molecular techniques such as Western blot, qRT-PCR, RNA-binding protein immunoprecipitation and Dual-Luciferase reporter assays were implemented to investigate the role of circ-0000658/miR-1227/interferon regulatory factor-2 (IRF2) axis in OS. Eventually, the impact of circ-0000658 on tumour growth and metastasis was observed in a xenograft mouse model. The results of this study revealed that circ-0000658 exhibits low levels in OS tissues and cell lines. Moreover, circ-0000658 repression promoted cell cycle, proliferation, invasion and migration but inhibited the apoptosis of OS cells. Researches have previously shown that circ-0000658 contains a binding site for miR-1227 and thus can abundantly sponge miR-1227 to up-regulate the expression of its target gene IRF2. Moreover, both inhibition of miR-1227 and overexpression of IRF2 reversed cell proliferation and invasion, which was triggered by circ-0000658 repression. Conclusively, circ-0000658 modulates biological function of OS cells through the miR-1227/IRF2 axis. Therefore, circ-0000658 might act as a possible novel therapeutic target for the treatment of OS.

Keywords: circ-0000658; IRF2; circRNA; miR-1227; osteosarcoma.

FIGURE 1

Circ‐0000658 expression level is reduced in OS tissues and cell lines. The microarray GSE96964 in the platform GPL19978 containing seven human OS cell lines (U2OS, MTX300, HOS, MG63, X143B, ZOS and ZOSM) and the human osteoblast hFOB1.19 was utilized for this analysis. (A) Heatmap of circRNA microarray is presented here. (B) Resistance of circ‐0000658 in OS cells to RNase R digestion is presented here. (C, D) The expression of circ‐0000658 in OS tissues and cell lines. (E) High circ‐0000658 expression level is related to a longer overall survival. Data represent the mean ± SD of 3 independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001

FIGURE 2

Circ‐0000658 restricts OS cell proliferation and cell cycle. (A) Circ‐0000658 expression in MG63 and HOS cells after transfection. (B) CCK‐8 assay, (C) EdU (bar = 100μm) and (D) colony formation assays were performed to figure out the influence of circ‐0000658 on cell proliferation. (E) The cell cycle of MG63 and HOS cells after transfection. Data represent the mean ± SD of 3 independent experiments; **P < 0.01, ***P < 0.001

FIGURE 3

Circ‐0000658 up‐regulation promotes OS cell apoptosis, but inhibits cell invasion. A, Increase in circ‐0000658 level aggravates OS cell apoptosis showed by apoptosis assay. B, Increase in circ‐0000658 level weakens the ability of OS cells to invade showed by transwell assay (bar = 100 μm). Data represent the mean ± SD of 3 independent experiments; **P < 0.01, ***P < 0.001

FIGURE 4

Mutual repression between circ‐0000658 and miR‐1227. A, qRT‐PCR assay results revealed that the expression level of miR‐1227 is increased in OS tissues. B, Correlation between miR‐1227 and circ‐0000658 in OS samples. C, The binding sites between miR‐1227 and circ‐0000658, and Dual‐Luciferase reporter assay. D, RIP assay in HOS and MG63 cells. E, Circ‐0000658 inversely modulates miR‐1227 expression. Data represent the mean ± SD of 3 independent experiments; **P < 0.01, ***P < 0.001

FIGURE 5

Circ‐0000658 regulates IRF2, which is the target gene of miR‐1227. A, The binding sites between miR‐1227 and IRF2. B, Dual‐Luciferase reporter assay in 293T cells. C, IRF2 expression in OS tissues. D, Correlation between miR‐1227 and IRF2 in OS samples. E, Correlation between IRF2 and circ‐0000658 in OS samples. F, G, qRT‐PCR and Western blot analyses results revealed that miR‐1227 represses IRF2 expression in OS cells. H, I, Anti‐miR‐1227 treatment reversed the effect of circ‐0000658 overexpression on IRF2 expression showed by qRT‐PCR and Western blot assays. Data represent the mean ± SD of 3 independent experiments; **P < 0.01, ***P < 0.001, ns: no statistical significance

FIGURE 6

Circ‐0000658 limits the tumour growth and metastasis in the body. A, Xenograft tumours. B, The growth of xenograft tumours from circ‐0000658 cells is slower than that of xenograft tumours from Lv‐NC cells. C, The mean weight of xenograft tumours. D, Circ‐0000658 expression in xenograft tumours was determined. E, Circ‐0000658 overexpression markedly induces IRF2 in tumours compared with negative control group (bar = 50 μm). F, Up‐regulation of circ‐0000658 inhibits tumour metastasis in vivo. Representative macroscopic and microscopic images (H&amp;E staining) of the lungs. G, Up‐regulation of circ‐0000658 promotes tumour apoptosis (bar = 20 μm). **P < 0.01, ***P < 0.001

FIGURE 7

Circ‐0000658 modulates OS cell proliferation and invasion through miR‐1227/IRF2 axis. (A) EdU (bar = 100 μm) and (B) colony formation assays were used to assess the cell proliferation of MG63 and HOS cells. (C) Representative images of invasion assay of MG63 and HOS cells (bar = 100 μm). Data represent the mean ± SD of 3 independent experiments; **P < 0.01, ***P < 0.001