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.