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.