Circ_0001722 acts as a sponge for miR-204-5p.
The sequences of circRNAs are highly conservative, and circRNAs can
function as miRNA sponge to regulate gene expression.
Through bioinformatical analysis,
we discovered that complementary pairing sites (80-89 nt) on
circ_0001722 that could bind to miR-204-5p (Figure 3a). To validate
binding capability of the miR-204-5p to circ_0001722, we constructed
the circ_0001722 luciferase reporter system. In the dual-luciferase
reporter assay, comparing with miR-NC, miR-204-5p mimics could
significantly suppressed the luciferase activity of pmirGLO-Wt-circ in
HEK293T cells, while the luciferase activity of pmirGLO-Mt-circ could
not be suppressed (Figure 3b). We next performed Ago2
immunoprecipitation to determine whether circ_0001722 served as a
platform for Ago2 and miR-204-5p. As shown in Figure 3c, circ_0001722
and miR-204-5p were abundantly enriched more in Ago2 protein than in
IgG, suggesting that expression of miR-204-5p could be affected by
circ_0001722. Moreover, the expression of miR-204-5p was elevated in
circ_0001722 silenced MG63 and U2OS cells (Figure 3d). Furthermore, the
results of Pearson’s correlation analysis showed the circ_0001722 level
was inversely to correlate with miR-204-5p level in 20 human OS tissues
(Figure 3e), which providing evidence of the potential correlation
between circ_0001722 and miR-204-5p. Given all of these data,
circ_0001722 not only targeted miR-204-5p but also acted as a sponge
for miR-204-5p in OS cells.