Research Paper Volume 15, Issue 5 pp 1564—1590

Figure 3. circRBMS3 serves as a sponge for miR-424 in OS cells. (A) AGO2 RNA immunoprecipitation assay for circRBMS3 levels in 143B cells stably expressing shcircRBMS3. Data represent the mean ± SD for three experiments. * P < 0.05. (B) Schematic illustration showing overlapping of the target miRNAs of circRBMS3 predicted by miRanda, Targetscan, and RNAhybrid. (C, D) Lysates prepared from HOS and 143B cells were subjected to an RNA pull-down assay and tested by (C) RT–qPCR and (D) qPCR. Relative levels of circRBMS3 were normalized to input. Data represent the mean ± SD (n = 3). * P < 0.05 versus oligo probe (Student’s t-test). (E) The relative levels of 11 miRNA candidates in HOS and 143B cell lysates, as detected by qPCR and RT–qPCR. (F) Schematic illustration demonstrating complementary miR seed sequence with circRBMS3. Lowercase letters indicate mutated nucleotides. (G) 293T cells were co-transfected with miR mimics and a luciferase reporter construct containing wild-type (WT) or mutated (MUT) circRBMS3. Data represent the mean ± SD (n = 3). * P < 0.05. (H) FISH images showing co-localization of circRBMS3 and miR-424 in 143B cells. CircRBMS3 probes were labeled with Alexa Fluor 488. Locked nucleic acid miR-424 probes were labeled with Cy3. Nuclei were stained with DAPI. Scale bar, 50 μm.