Research Paper Volume 12, Issue 8 pp 6644—6666

Figure 5. miR-142 and miR-22 are inhibited by Dox treatment and negatively correlated with Sox2OT-V7. (A, B) The expression of miR-142 and miR-22 in chemosensitive and chemoresistant OS tissues was determined by qPCR. (C, D) The correlation of Sox2OT-V7, miR-142, and miR-22 was analyzed by Pearson’s correlation analyses. (E) miR-142 and miR-22 expression in OS cells treated with Dox was determined by qPCR. (F, G) miR-142 and miR-22 overexpression and inhibition in U2OS cells were achieved by transfection of miR-142 and miR-22 mimics or inhibitor, as confirmed by qPCR. (H) miR-142 and miR-22 expression in Sox2OT-V7 silenced OS cells was determined by qPCR. (I) Predicted miR-142 and miR-22 binding sites in Sox2OT-V7. Wild-type and mutant-type Sox2OT-V7 reporter vectors containing wild-type or mutant-type miR-142 or miR-22 binding sites were constructed. (J) The above-described vectors were cotransfected in HEK293 cells with miR-142 or miR-22 mimics or inhibitor, and the luciferase activity was determined. (K) Association of Sox2OT-V7, miR-142, and miR-22 with AGO2 in HEK293 cells. Detection of AGO2 and IgG using immunoblotting assays. (L, M) RIP assay in HEK293 cells transfected with control miRNA (miR-NC) or miR-142 mimics or miR-22 mimics followed by real-time PCR to detect Sox2OT-V7 associated with AGO2. The data are presented as mean ± SD of three independent experiments. *P<0.05, **P<0.01.