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. 2017 Sep 27;8(54):92454–92469. doi: 10.18632/oncotarget.21384

Figure 2. MiR-34a downregulates PLCE1 expression by directly targeting its 3′-UTR.

Figure 2

(A) Prediction of upstream miRNAs targeting PLCE1 by online miRNA target prediction databases (TargetScan, miRanda, and miRDB). (B) List of the putative miR-34a binding sequences in the 3′-UTR of PLCE1. Mutations were generated in the PLCE1 3′-UTR sequence in complementary sites for seed regions in miR-34a. A human PLCE1 3′-UTR fragment containing either the wild type or mutant miR-34a binding sequence was cloned downstream of the luciferase reporter gene. (C) Analysis of luciferase activity. Eca109 cells were co-transfected with the Renilla luciferase expression construct psiCHECK-2 as the internal control, a firefly luciferase reporter plasmid containing either the wild type or mutant PLCE1 3′-UTR, and either the miR-34a mimic or the negative control (NC). Firefly luciferase activity was normalized to Renilla luciferase activity. Data were from at least three independent experiments. * P < 0.05 and ** P < 0.01 compared with controls. (D-E) Negative regulation of PLCE1 protein expression by miR-34a. Endogenous PLCE1 protein and miR-34a levels in Eca109 cells transfected with miR-34a mimic or NC, and in TE-1cells transfected with miR-34a inhibitor or NC, were analyzed by Western blot and real-time PCR after 70 h transfection.