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. 2012 Sep 19;7(9):e45133. doi: 10.1371/journal.pone.0045133

Figure 3. Regulation of P21 and BIM by the miR-106b-93-25 cluster.

Figure 3

(a) pGL3 luciferase reporter constructs containing either the wild-type or mutant 3′UTR target sequence of miR-106b or miR-25 in the P21 or BIM gene were co-transfected into ECC-1 cells with either miRNA-negative control, miRNA mimics or empty pGL3 control vector (each n = 3). Luciferase activity was determined in the cell extracts after 24 h. In the presence of the wild-type P21 3′UTR, the miR-106b mimics significantly inhibited the luciferase activity compared with vector control. This inhibition was not observed with the mutant 3′UTR reporter construct (left). In the presence of the wild-type BCL2L11 3′UTR, the miR-25 significantly inhibited the luciferase activity compared with vector control. This inhibition was attenuated with the mutant 3′UTR reporter construct (right). (b) qRT-PCR analysis of P21 and BIM mRNA levels after the transfection of ECC-1 cells with miR-106b or miR-25 mimics. P21 mRNA was significantly decreased by miR-106b mimics while BIM mRNA was not significantly changed by miR-25 mimics. (c) Western blot analysis showing downregulated P21 and BIM proteins in ECC-1 and HEC-1A cells transfected with miR-106b and miR-25 mimics. (d) qRT-PCR and Western blot analysis showing upregulated P21 and BIM mRNA and protein level in ECC-1 and HEC-1A cells treated with TSA for 24 h. **, P<0.01; *, P<0.05, paired t-test.