Research Paper Volume 15, Issue 5 pp 1564—1590

Figure 5. EIF4B and YRDC are direct targets of miR-424. (A) Volcano plots of protein profiles. 143B Cells were transfected with shcircRBMS3, followed by protein profiles, with 3 repeats for each sample. (B) KEGG analysis of protein profiles. 143B cells were transfected with shcircRBMS3, followed by protein profiles, with 3 repeats for each sample. (C) Schematic illustration showing overlapping of the target mRNAs of circRBMS3 and miR-424 predicted by RNA-seq, Targetscan, and by Mass Spec analysis. (D) Heatmap of differentially expressed proteins after circRBMS3 knockdown. (E) EIF4B and YRDC expression levels were higher in human OS than in chondroma tissue examined by T test. Data represent the mean ± SD (n = 12). (F) EIF4B and YRDC expression levels were higher in human OS than in chondroma tissue. Representative images are shown. (G) Kaplan-Meier survival analysis of EIF4B and YRDC low and high sarcoma patients (log rank test). (H) Schematic illustration showing complementarity to the miR-424 seed sequence in the 3’-UTR of EIF4B and YRDC. Lowercase letters indicate mutated nucleotides. (I) 293T cells were co-transfected with pre-miR-424 and luciferase reporter constructs containing wild-type (WT) or mutated EIF4B and YRDC 3’-UTRs. Data represent the mean ± SD (n = 3). * P < 0.05. (J–L) MiR-424 overexpression reduced EIF4B and YRDC (J, K) protein and (L) mRNA levels while miR-424 inhibition increased EIF4B and YRDC (J, K) protein and (L) mRNA levels. Cells were transfected with NC or miR-424 mimic/inhibitor, and mRNA or protein levels evaluated. Protein expression was evaluated by western blot and immunofluorescence; mRNA levels were evaluated by RT-qPCR. Data represent the mean ± SD (n = 3). * P < 0.05. Scale bars = 50 μm.