Fig. 4

miR-506 overexpression downregulates SP1, SP3, DNMT1 and upregulates MEG3 expression. a RT-qPCR analysis showing the level of miR-506 in control (wild-type), mimic NC- or miR-506 mimic transfected MCF-7 cells. b Western blot analysis showing the protein level of DNMT1 and SP3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. β-actin acts as internal control. c RT-qPCR analysis showing the mRNA level of DNMT1 and SP3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as internal control. d Bioinformatic prediction of binding site at 3′-UTR of SP1 by miR-506. e Luciferase reporter assay showing miR-506 binds wild-type 3′-UTR of SP1, not mutant 3′-UTR of SP1. The relative luciferase activity was measured and the data were presented as mean ± SD. f Bioinformatic prediction of binding site at 3′-UTR of SP3 by miR-506. g Luciferase reporter assay showing miR-506 binds wild-type 3′-UTR of SP3, not mutant 3′-UTR of SP3. The relative luciferase activity was measured and the data were presented as mean ± SD. h RT-qPCR analysis showing the level of MEG3 in control (wild-type), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as internal control. i Methylation-specific PCR (MSP) analysis showing the methylation level of MEG3 promoter in wild-type (control), mimic NC- or miR-506 mimic-overexpressed MCF-7 cells. GAPDH acts as negative control. j Methylation-specific PCR (MSP) analysis showing the methylation level of MEG3 promoter in wild-type (control), mimic NC- or miR-506 mimic-overexpressed MDA-MB-231 cells. GAPDH acts as negative control. All data were presented as mean ± SD from three biological replicates (*P < 0.05; **P < 0.01)