Figure 5.

miR-124 Regulation by MEG3 via p53

(A) Western blot analysis of the protein levels of p53 in the ACHN and 786-O cells transfected with lenti-MEG3 or miR-124 mimics and inhibitors. β-Actin served as an internal control. (B) RIP was performed using the p53 antibody to immunoprecipitate MEG3 and a primer to detect MEG3 (left, ACHN cells; right, 786-O cells). Ab, p53 antibody; IgG, an antibody control; M, Marker; tRNA, total RNA, as positive control. (C) MEG3 relative level was measured after the p53 antibody immunoprecipitating MEG3. **p < 0.01. (D) A schematic of the luciferase vectors with miR-124 promoter fragments and sequence information for the p53 binding site mutations. Sequence alignment of predicated putative p53 binding site in the promoter of miR-124. Schematic diagram of our constructed miR-124 luciferase reporter plasmids. (E) ChIP assay were performed using the p53 antibody to immunoprecipitate the promoters of miR-124 promoters in wild-type p53 or p53 knockdown cells. (F) Luciferase assay using the DNA sequence of miR-124 promoters with wild-type (WT) or mutant (mut) p53-binding sites under p53 knockdown. (G) Inhibition of p53 protein expression by siRNAs to p53 (sip53-1 and sip53-2). ACHN and 786-O cells were transfected with sip53 or siRNA control as indicated. After 48 h, p53 and internal control β-actin were detected by western blot. (H) Expression levels of miR-124 in ACHN and 786-O cells harboring wild-type p53, and p53 knockdown using siRNA. (I) Expression levels of miR-124 in ACHN and 786-O cells after transfection with lenti-MEG3.