Fig 4. TP53 is a direct miR-34a target.
(A) TP53 mRNA is enriched in Bi-miR-34a PDs in HCT116 cells. mRNA levels were determined by qRT-PCR and plotted as fold change relative to mRNAs pulled down with the Bi-control miRNA (Bi-ctl-miRNA). The housekeeping genes UBC and SDHA were used as negative controls. An additional control was PD of unbiotinylated miR-34a. (B) miR-34a does not affect the activity of a luciferase reporter containing the full length 3’UTR of TP53. A reporter containing the 3’UTR of MYB was used as positive control. Luciferase activity is relative to cells transfected with the control miRNA. (C) Pairing of miR-34a to predicted TP53 MREs. The miR-34a seed region is highlighted in blue, while mutations (mt) introduced in the MREs are highlighted in red. Black dashes indicate Watson-Crick base pairing and red dashes G:U base pairing. The numbers in parentheses indicate the position of the MRE in the mRNA. (D) miR-34a recognition of predicted wild-type (wt) or mutant (mt) TP53 miR-34a MREs cloned into the 3’UTR of Renilla luciferase were assessed in dual luciferase reporter assays in cells transfected with miR-34a relative to cells transfected with control miRNA. (E,F) The function of predicted TP53 miR-34a MREs (wt or mt) in their native location in full length TP53 mRNA was assessed in TP53 -/- HCT116 cells cotransfected with control miRNA or miR-34a mimics and wt or mt p53 cDNA. p53 protein was analyzed by p53 vs β-actin immunoblot 48 hr later. A representative blot is shown in (E) and densitometry of p53 relative to β-actin signal in 3 independent experiments in cells transfected with miR-34a relative to cells transfected with control miRNA is shown in (F). (G) miR-34a over-expression in p53-proficient HCT116 cells increases p53 protein. WT HCT116 cells were transfected with control or miR-34a mimics and protein levels were analyzed by immunoblot 48 hr post-transfection. CDK6 and β-actin immunoblots are shown as controls. (H) qRT-PCR analysis of CDK6, TP53 and CDKN1A mRNA in samples from (G). Levels are normalized to expression in control (ctl) miRNA-transfected cells. (I) miR-34a overexpression in HCT116 cells increases p53 protein stability. Pulse-chase analysis of p53 protein in HCT116 cells transfected with control or miR-34a mimics. DOX-treated HCT116 cells are a positive control. All graphs show the mean +/- STDEV of at least three independent experiments (*, p<0.05; **, p<0.01, relative to control miRNA-transfected cells, 2-tailed Student’s t-test). Immunoblots are representative of at least 3 independent experiments.