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. 2016 Sep 27;7(43):70857–70868. doi: 10.18632/oncotarget.12268

Figure 3. P70S6K1 is a direct target of miR-145.

Figure 3

A. Schematic diagram of putative miR-145 binding site in the 3′-UTR region of p70S6K1 in human. The seed sequence of miR-145 matches 3'-UTR regions of p70S6K1 (in bold). The mutated nucleotides of the p70S6K1 3'-UTR were labeled in red. B. HEK-293T cells were co-transfected with control (miR-NC) or pre-miR-145 mimics along with WT or mutant p70S6K1 luciferase reporter as indicated. Luciferase activities were measured 24 h post transfection using the dual-luciferase reporter assay system. C. Cells were treated with different doses of gemcitabine (0, 1.5 and 2.5 μM), the expression levels of p70S6K1, p-S6 and HIF-1α were determined by Western blotting. GAPDH is used as an internal control. D. Cells were transfected with miR-145 or miR-NC. After 72 h, the expression levels of p70S6K1, p-S6, HIF-1α and VEGF were determined as above. E. VEGF levels were measured by RT-qPCR in cell lines stably overexpressing miR-NC and miR-145, and normalized to level of GAPDH. F: Spearman′s correlation analysis was used to detect the correlation between expression levels of p70S6K1 and miR-145 at mRNA level in human pancreatic tumor specimens. Data represent mean ± SEM of three replicates. * indicated significant difference at P<0.05;** indicated significant difference at P<0.01.