Figure 5.

(a) U2OS cells were transfected with shHOXA-AS2 #2 for 48 hrs. Hierarchical clustering revealed systematic variations in the expression of miRNAs. Numerous differentially expressed miRNAs between control and shHOXA-AS2 #2 transfected U2OS cells are shown on a scale from green (low) to red (high); (b) StarBase v2.0 results showing the sequence of HOXA-AS2 with highly conserved putative miR-520c-3p binding sites; (c) Expression levels of miR-520c-3p in different OS cell lines were determined by qRT-PCR; (d) Silencing of HOXA-AS2 increased the expression level of miR-520c-3p in U2OS and MG-63 cells; (e) miR-520c-3p inhibited the expression of HOXA-AS2 in U2OS and MG-63 cells; (f) The wild-type or mutant miR-520c-3p-binding sites in HOXA-AS2 were inserted into pMIR-report luciferase vector. Luciferase activity was detected in OS cells co-transfected with miR-520c-3p or negative control (miR-control) and reporter plasmids containing WT-HOXA-AS2 (wild type) or MUT-HOXA-AS2 (mutant type). The normalized luciferase activity in the miR-control group was used as the relative luciferase activity. All tests were at least performed three times. Data were expressed as mean ± SD. *P < 0.05, **P < 0.01.