Figure 5
Regulatory feedback between miR-29b and its target Sp1. (a) Dual-luciferase assay of SKMM1 and NCI-H929 cells co-transfected with firefly luciferase constructs containing the 3′UTR of Sp1 (nts 2503–5200) and miR-29b or scrambled oligonucleotides (NC) as indicated. The firefly luciferase activity was normalized to renilla luciferase activity. The data are shown as relative luciferase activity of miR-29b-transfected cells as compared with the control (NC) of a total of eight experiments from two independent transfections. (b) Quantitative RT–PCR of Sp1 24 h after transfection with synthetic miR-29b or scrambled oligonucleotides (NC) of SKMM1 and NCI-H929 cells. The results are shown as average mRNA expression after normalization with GAPDH and ΔΔCt calculations. (c) Immunoblot of Sp1 24 h after transfection with synthetic miR-29b or scrambled oligonucleotides in SKMM1 and NCI-H929 cells. The protein loading control was performed using GAPDH. (d) Quantitative RT–PCR of Sp1 in SKMM1 cells transduced with the empty vector or antagomiR-29b (anti-miR-29b). The results are shown as average mRNA expression after normalization with GAPDH and ΔΔCt calculations. (e) Dual-luciferase assay of SKMM1 and NCI-H929 cells transfected with a firefly luciferase construct containing the miR-29a/b1 promoter together with an Sp1 expression construct or pBABE empty vector as control. *P<0.001. (f) Quantitative RT–PCR of miR-29b in SKMM1 and NCI-H929 cells 24 h after transfection with the empty vector (pBABE) or the Sp1-expression vector. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values. (g) Quantitative RT–PCR of miR-29b in SKMM1 cells stably expressing two different shRNAs against Sp1 (shSp1#1 and shSp1#2) or a scrambled sequence (SCR). The immunoblot shows the levels of Sp1 after silencing by Sp1-specific shRNAs. GAPDH was used as loading control. (h) Quantitative RT–PCR of miR-29b in SKMM1 cells treated with 160 nM mithramycin-A or vehicle (DMSO) for 15 h. *P<0.001; •P<0.05
