Figure 8. Validation of potential miRNA targets.

(A) BRL and RH-35 cells were transfected with miR-106b, 93, and 25, and the mimic control was used as a control. After 48 hours, the potential target expression was monitored by real-time PCR and was represented as the expression level relative to the control. The data represent at least 3 independent experiments with SEM. *P < 0.05. (B) Schematic view of the miRNA binding sites in the 3′ UTRs of the RB1, KAT2B, and CEBPA genes and their corresponding mutated sites, which were constructed in a luciferase system. (C) HEK293T cells were cotransfected with 10 nmol/L miRNA mimics or with the control and different pMIR-constructs. After 48 hours, the cells were harvested and the luciferase activities analyzed. All Renilla luciferase activities were normalized against the firefly luciferase activity. The data represent at least 3 independent experiments with SEM. *P < 0.05. (D) miR-106b and miR-93 decreased the protein expression of RB1, and miR-25 decreased KAT2B as assessed by western blot analysis. Western bloting analyses of RB1 and KAT2B in BRL and RH-35 cells transfected miRNA mimics or inhibitors, actin was used as the loading control. (E) The representative expression of RB1 in the tissues of animals by western blotting at 24 hours after PH. RB1 was up-regulated by the spong for miR-106~25, and inhibited by miR-106~25.