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. 2012 Feb 15;11(4):785–796. doi: 10.4161/cc.11.4.19228

Figure 5.

Figure 5

Suppressing the expression of miR-205 compromises the IR-induced downregulation of PTEN. (A) MiR-205 represses PTEN expression by targeting the 3′-UTR of PTEN mRNA. An miR-205 binding site within PTEN 3′UTR that is conserved in different species was predicted by TargetScan. The “seed” sequences of miR-205 complementary to PTEN are shown in red box. (B) Hybridization of miR-205 (green) and PTEN mRNA (red) was predicted by RNAhybrid software. The minimum free energy (mfe) required for this hybridization is indicated. (C) Expression of Mir 205 in CNE-2 cells leads to downregulation of PTEN and increased akt activity. Levels of PTEN protein expression and phosphorylation of Akt after transfection of miR-205 into CNE-2 cells were demonstrated by indicated antibodies. (D) PTEN mRNA levels is downregulated after miR-205 transfection. Gene expression was measured by qRT-PCR. Data were presented as mean ± SEM (n = 3). ****p < 0.0001. (E) Reporter gene assay. pMIR-REPORT, 3′-PTEN-miR-205 and the mutant reporter 3′-PTEN-miR-205-deletion were transfected into CNE-2/vector and CNE-2/miR-205 cells. Luciferase reporter gene activities were assayed. Data were presented as mean ± SEM of 3 experiments. ***p < 0.001. (F) Suppressing the expression of Mir-205 compromises the IR-induced downregulation of PTEN. Indicated cells were irradiated with 10 Gy IR or without. The lysates were immunoblotted with indicated antibodies.