Figure 5. Validation of STAT3 mRNA as a direct target of miR-769-5p in RB cells. (A) The wild-type miR-769-5p–binding site in the 3′-UTR of STAT3 mRNA was predicted by bioinformatic analysis. The mutant binding site is also shown. (B) The luciferase reporter plasmid harboring either the wild-type or mutant miR-769-5p–binding site was cotransfected with either agomir-769-5p or agomir-NC into Y79 and WERI-RB-1 cells. The Dual-Luciferase Reporter Assay System was used to detect the Firefly luciferase activity by normalizing it to the activity of Renilla luciferase. *P < 0.05 vs. group agomir-NC. (C) RT-qPCR was carried out to quantitate STAT3 mRNA in the 47 RB tissue samples and 13 normal retinal tissue samples. *P < 0.05 vs. normal retinal tissue samples. (D) Spearman’s correlation analysis was performed to study the correlation between the expression of miR-769-5p and STAT3 mRNA among the 47 RB tissue samples; R2 = 0.3020, P < 0.0001. (E, F) Y79 and WERI-RB-1 cells were transfected with either agomir-769-5p or agomir-NC. The mRNA and protein levels of STAT3 were measured by RT-qPCR and western blotting, respectively. *P < 0.05 vs. the agomir-NC group.