Sh-circ_0001722 inhibits the proliferation and invasion of human OS cells by upregulating miR-204-5p.
In order to further investigate whether circ_0001722 plays a promotion role in OS cells through binding with miR-204-5p, we conducted the following experiments. We transfected MG63 and U2OS cells with miR-204-5p, making miR-204-5p expression increased remarkably, which also could be observed after sh-circ_0001722 transfection as well (Figure 4a). The cell proliferation assays showed that both upregulation of miR-204-5p and downregulation of circ_0001722 could suppress OS cell proliferation ability in vitro (Figure 4b). Transwell assay showed that cell invasion was remarkably restrained after sh-circ_0001722 or miR-204-5p transfection (Figure 4c). These findings demonstrated that miR-204-5p contributed to proliferation and invasion of OS cells, and sh-circ_0001722 reduced tumorigenesis in OS cells by directly binding and upregulating miR-204-5p.
Sh-circ_0001722 mediates OS cells suppression through the miR-204-5p /RUNX2 axis.
Computational algorithms predicted that miR-204-5p could specifically bind to complementary sequence (268-274 nt) of RUNX2 3’UTR (Figure 5a). We carried out the 3’UTR luciferase reporter assay to verify whether miR-204-5p can bind to RUNX2 3’UTR and inhibit its expression. The result showed that miR-204-5p could significantly reduce the luciferase activity of pmirGLO-Wt-3’UTR clone, but could not affect the luciferase activity of pmirGLO-Mt-3’UTR clone (Figure 5b), indicating that miR-204-5p can directly target the 3’UTR of RUNX2 mRNA, leading to the inhibition of its translation. In addition, Western blotting analysis showed that, both miR-204-5p and sh-circ_0001722 could induce a significantly reduction of endogenous RUNX2 expression in OS cells (Figure 5c). Moreover, rescue assays showed that the cell proliferation ability inhibited by sh-circ_0001722 could be rescued by exogenous RUNX2 (Figure 5d). The inhibitory effect of sh-circ_0001722 on cell invasion could also be rescued by exogenous RUNX2 as well (Figure 5e). Our results verified that miR-204-5p contributed to proliferation and invasion of OS cells through targeting RUNX2 mRNA, and sh-circ_0001722 reduced tumorigenesis of OS cells by directly binding and upregulating miR-204-5p/RUNX2 axis.