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. 2019 Jan 15;16:91–104. doi: 10.1016/j.omtn.2019.01.004

Figure 5.

Figure 5

miR-582-3p and miR-582-5p Target Several Components of TGF-β Signaling

(A) Real-time PCR analysis of SMAD2, SMAD4, TGFBRI, and TGFBRII expression in the pri-miR-582-transfecting or vector PCa cells. Transcript levels were normalized by GAPDH expression. Error bars represent the mean ± SD of three independent experiments. *p < 0.05. (B) Western blotting of SMAD2, SMAD4, TGFBRI, and TGFBRII expression in the pri-miR-582-transfecting or vector PCa cells. α-Tubulin served as the loading control. (C) Luciferase assay of the cells transfected with pmirGLO-3′ UTR reporter of SMAD2, SMAD4, TGFBRI, and TGFBRII in the pri-miR-582-transfecting or vector PC-3 cells. Error bars represent the mean ± SD of three independent experiments. *p < 0.05. (D) Microribunocleoprotein (miRNP) IP assay showing the association between miR-582-3p and miR-582-5p and SMAD2, SMAD4, TGFBRI, and TGFBRII transcripts in the pri-miR-582-transfecting or vector PC-3 cells. Pulldown of IgG antibody served as the negative control. Error bars represent the mean ± SD of three independent experiments. *p < 0.05. (E–G) Upregulating TGFBRI or SMAD2 partially rescued the (E) TGF-β signaling activity, (F) invasion, and (G) migration abilities in the pri-miR-582-transfecting PCa cells in the presence of TGF-β (5 ng/mL). *p < 0.05; **p < 0.01.