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. 2019 Feb 7;16:51–62. doi: 10.1016/j.omtn.2019.01.014

Figure 6.

Figure 6

PHLPP2 Was Upregulated at the Translational Level through Upregulated 3′ UTR Activity

(A) RT-PCR was used to analyze the mRNA level of PHLPP2 in T24T(Vector) and T24T(MEG3) cells. GAPDH was used as the mRNA loading control. (B) Diagram of various lengths of PHLPP2 3′ UTR luciferase reporters, including P1, P2, and P3. Arrows show the potential miR-27a binding sites. (C) The PHLPP2 mRNA 3′ UTR luciferase reporters, P1, P2, and P3 were co-transfected into T24T(Vector) and T24T(MEG3) cells with TK at a ratio of 10:1. After 24 h, the luciferase activity was determined. TK was used as the internal control. The asterisk indicates a significant difference compared with the vector transfectant (p < 0.05). The bars represent the mean ± SD of triplicates. (D) miR-27a expression was analyzed with qRT-PCR. GAPDH mRNA was used as the loading control. Results are presented as relative miR-27a levels. The asterisk indicates a significant difference compared with the vector transfectant (p < 0.05). The bars represent the mean ± SD of triplicates. (E) The cell extracts from the transfectants indicated were subjected to western blot analysis. GAPDH was used as the internal control. (F) The c-Myc promoter-driven luciferase reporter, together with the internal control TK reporter was transiently transfected into UMUC3(MEG3/Vector) and UMUC3(MEG3/miR-27a) cells. Twenty-four hours after transfection, the cells were extracted for luciferase assay. The asterisk indicates a significant increase compared with the vector transfectant (p < 0.05). The bars represent the mean ± SD of triplicates. (G) The PHLPP2 3′ UTR luciferase reporter P1 was transiently transfected into UMUC3(MEG3/Vector) and UMUC3(MEG3/miR-27a) cells, together with its internal control construct TK. (H) PHLPP2 3′-UTR luciferase reporter P1 and its point mutants (as indicated) were transiently transfected into T24T(Vector) and T24T(MEG3) cells, together with its internal control construct TK. Twenty-four hours after transfection, the cells were extracted for a luciferase assay. The asterisk indicates a significant inhibition compared with the vector transfectant (p < 0.05). The bars represent the mean ± SD of triplicates.