CircDOCK1 promotes the tumorigenesis and cisplatin resistance of osteogenic sarcoma via the miR-339-3p/IGF1R axis

Abstract

Background: Circular RNAs (circRNAs), a class of noncoding RNAs (ncRNAs), may modulate gene expression by binding to miRNAs. Additionally, recent studies show that circRNAs participate in some pathological processes. However, there is a large gap in the knowledge about circDOCK1 expression and its biological functions in osteogenic sarcoma (OS).

Methods: Differentially expressed circRNAs in OS cell lines and tissues were identified by circRNA microarray analysis and quantitative real-time PCR (qRT-PCR). To explore the actions of circDOCK1 in vivo and in vitro, circDOCK1 was knocked down or overexpressed. To assess the binding and regulatory associations among miR-339-3p, circDOCK1 and IGF1R, we performed rescue experiments, RNA immunoprecipitation (RIP), RNA pulldown assays and dual-luciferase assays. Moreover, we performed apoptosis assays to reveal the regulatory effects of the circDOCK1/miR-339-3p/IGF1R axis on cisplatin sensitivity.

Results: CircDOCK1 expression remained stable in the cytoplasm and was higher in OS tissues and cells than in the corresponding controls. Overexpression of circDOCK1 increased oncogenicity in vivo and malignant transformation in vitro. In the U2OS and MG63 cell lines, circDOCK1 modulated tumor progression by regulating IGF1R through sponging of miR-339-3p. Additionally, in the U2OS/DDP and MG63/DDP cell lines, cisplatin sensitivity was regulated by circDOCK1 via the miR-339-3p/IGF1R axis.

Conclusions: CircDOCK1 can promote progression and regulate cisplatin sensitivity in OS via the miR-339-3p/IGF1R axis. Thus, the circDOCK1/miR-339-3p/IGF1R axis may be a key mechanism and therapeutic target in OS.

Keywords: Cisplatin resistance; IGF1R; OS; circDOCK1; miR-339-3p.

Fig. 1

The expression and characterization of circDOCK1 in OS cells and tissues. A circDOCK1 expression in OS tissues was measured by qRT–PCR. B The association of circDOCK1 expression with overall survival in 70 OS patients was evaluated by Kaplan–Meier analysis. C Relative serum circDOCK1 levels in OS patients and non-OS carriers (n = 70). D Diagnostic value of serum circDOCK1 by ROC curve. E CircDOCK1 expression in OS cells (normalized to hFOB 1.19 cells). F Sanger sequencing of circDOCK1, with the arrows indicating splice sites. G After treatment with RNase R, the abundance of circDOCK1 and linear DOCK1 in U2OS and MG63 cell lines was determined by qRT–PCR (normalized to Mock). H qRT-PCR products of linear and circular products amplified with convergent and divergent primers with and without RNase R treatment. I qRT–PCR analysis of circDOCK1 and DOCK1 expression in OS cells after actinomycin D treatment. J qRT–PCR analysis of isolated cytoplasmic and nuclear fractions. K FISH assays using circDOCK1 and negative control probes in the MG63 and U2OS cell lines (scale bar = 10 μm). The data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns: nonsignificant

Fig. 2

CircDOCK1 promotes the malignant transformation of OS cells. A circDOCK1 RNA expression was measured by qRT–PCR post transfection of the overexpression plasmid, siRNA1 and siRNA2 (normalized to si-NC or vector). B CCK-8 assay assessing MG63 and U2OS cell proliferation post transfection with circDOCK1 siRNAs or the overexpression plasmid. C EdU incorporation assays assessing MG63 cell proliferation (scale bar = 100 μm). D Colony formation assays (normalized to si-NC or vector). E Flow cytometry detecting the influence of circDOCK1 on cell apoptosis. F-G Transwell assays detecting the influence of circDOCK1 on cell migration (F) and invasion (G) (normalized to si-NC or vector, scale bar = 100 μm). The data are presented as the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001, *vs. si-NC, ^#vs. vector

Fig. 3

CircDOCK1 serves as a miR-339-3p sponge. A Linear DOCK1 expression in OS tissues. B Association between circDOCK1 and DOCK1 (P = 0.207). C Suppression and overexpression of circDOCK1 had no modulatory effect on linear DOCK1 RNA expression in OS cells (normalized to si-NC or vector). D Suppression and overexpression of circDOCK1 had no modulatory effect on linear DOCK1 protein expression in OS cells. E Prediction of potential target miRNAs possibly binding to circDOCK1 with CircInteractome and starBase. F After pulldown with a circDOCK1-specific probe, miRNA expression was measured in lysates of MG63 and U2OS cells (normalized to the oligo probe). G PITA and TargetScan predictions of the target genes of miR-339-3p. H Combination of the prediction results with the GSE36001 RNA microarray analysis results. I Expression of potential targets of miR-339-3p in MG63 and U2OS cells post transfection with the miR-339-3p mimic or circDOCK1 siRNA (normalized to si-NC or miR-NC). J DOCK1 RNA expression was measured by qRT–PCR post transfection of the siRNA (normalized to si-NC). K RNA expression of IGF1R in MG63 and U2OS cells post transfection with the DOCK1 siRNA (normalized to si-NC). The data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns: nonsignificant

Fig. 4

Roles of circDOCK1 and IGF1R in vivo. A qRT–PCR detecting the mRNA level of IGF1R in OS tissues. B Western blot analysis measuring the protein level of IGF1R in OS tissues. C Positive association between circDOCK1 expression and IGF1R expression (P < 0.001). D circDOCK1 RNA expression was measured by qRT–PCR post transfection of the shRNA (normalized to sh-NC). E Subcutaneous injection of MG63 cells into nude mice. F Tumor volumes. G Tumor weights. H TUNEL assay (scale bar = 20 μm). I FISH of miR-339-3p and circDOCK1 in xenograft tumors (scale bar = 20 μm). J-K Luciferase signal intensities and H&E staining post tail vein injection in nude mice (scale bar = 100 μm). L Number of metastatic nodules in the lungs post tail vein injection. The data are presented as the mean ± SD values. **P < 0.01, ***P < 0.001

Fig. 5

CircDOCK1 regulates malignant transformation via the miR-339-3p/IGF1R axis in vitro. A Hypothetical and mutant binding sites in miR-339-3p for IGF1R (lower) and circDOCK1 (upper). B Luciferase reporter assay detecting the binding of miR-339-3p to circDOCK1 and IGF1R in MG63 cell lines (normalized to miR-NC). C RIP with an anti-AGO2 antibody in the MG63 cell line evaluating the transcript levels of circDOCK1 and miR-339-3p (left). Western blot analysis evaluating the AGO2 protein level (right) (normalized to a control). D-E The mRNA (D) and protein (E) levels of IGF1R in MG63 cell lines post transfection (normalized to IgG). F CCK-8 assay. G EdU incorporation assay (scale bar = 100 μm). H Flow cytometric analysis of apoptosis. I Transwell migration assay (normalized to miR-NC + vector, scale bar = 100 μm). J Transwell invasion assay (normalized to miR-NC + vector, scale bar = 100 μm). The data are presented as the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001

Fig. 6

Roles of IGF1R in vitro. A mRNA expression of IGF1R in cells post cotransfection with IGF1R siRNA and the miR-339-3p inhibitor (normalized to inh-NC + si-NC). B-F CCK-8 (B), EdU incorporation (scale bar = 100 μm) (C), flow cytometry (D), Transwell migration (normalized to inh-NC + si-NC, scale bar = 100 μm) (E) and Transwell invasion (normalized to inh-NC + si-NC, scale bar = 100 μm) (F) assays measuring the migration, proliferation, apoptosis and invasion of cells post cotransfection with IGF1R siRNA and the miR-339-3p inhibitor. The data are presented as the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001

Fig. 7

CircDOCK1 regulates the sensitivity of OS cells to cisplatin. A Relative viability of OS cells after 48 h of treatment with cisplatin at the specified concentrations. B The IC50 of OS cells treated with cisplatin. C The transcript levels of circDOCK1 and IGF1R in the hFOB 1.19, MG63, MG63/DDP, U2OS and U2OS/DDP cell lines (normalized to hFOB 1.19 cells). D Cell growth of MG63 cells and MG63/DDP cells after 48 h of treatment with cisplatin at the specified concentrations. E The IC50 of MG63 cells and MG63/DDP cells treated with cisplatin. F Cell apoptosis of MG63/DDP cells cotransfected with the miR-339-3p inhibitor and circDOCK1 siRNA. G Xenograft tumors from euthanized mice with or without cisplatin treatment. H Volumes of xenograft tumors. I The transcript levels of IGF1R and circDOCK1 in xenograft tumors (normalized to PBS + sh-NC). The data are presented as the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001