p53 represses c-Myc through induction of the tumor suppressor miR-145

Abstract

The tumor suppressor p53 negatively regulates a number of genes, including the proto-oncogene c-Myc, in addition to activating many other genes. One mechanism of the p53-mediated c-Myc repression may involve transcriptional regulation. However, it is not clear whether microRNAs (miRNAs) play a role in the p53-mediated posttranscriptional regulation of c-Myc. In this study, we show that a putative tumor suppressor, miR-145, is expressed through the phosphoinositide-3 kinase (PI-3K)/Akt and p53 pathways. Importantly, p53 transcriptionally induces the expression of miR-145 by interacting with a potential p53 response element (p53RE) in the miR-145 promoter. We further show that c-Myc is a direct target for miR-145. Although miR-145 silences the expression of c-Myc, anti-miR-145 enhances its expression. This specific silencing of c-Myc by miR-145 accounts at least in part for the miR-145-mediated inhibition of tumor cell growth both in vitro and in vivo. Finally, the blockade of miR-145 by anti-miR-145 is able to reverse the p53-mediated c-Myc repression. Together, these results define the role of miR-145 in the posttranscriptional regulation of c-Myc by p53 and suggest that, as a new member of the p53 regulatory network, miR-145 provides a direct link between p53 and c-Myc in this gene regulatory network.

Fig. 1.

Expression of miR-145 through the Akt and p53 pathways. (A) Expression of miR-145 in the matched breast and colon tumor specimens, respectively. In breast tumor tissue, the average level of miR-145 was 31% of that in the matched normal tissue; in colon tumor tissue, the corresponding number was 19%. (B) Induction of miR-145 by serum starvation. Cells (HCT-116 and HCT-8) were grown in the medium containing 10% or no FBS for 24 h. (C) Induction of miR-145 by PI-3K inhibitor LY294002. HCT-116 cells were treated with 50 μM LY294002 for 16 h. (D) Dominant negative Akt (Akt-DN) induces miR-145 expression. HCT-116 cells were transfected with vector alone (pCMV) or Akt-DN for 24 h. Values in B–D are means ± SE of 3 separate experiments and normalized to their respective controls as 1. **, P < 0.01.

Fig. 2.

Induction of miR-145 by p53. (A) MCF-7 cells were transduced with adenoviral pAd-GFP (Vector) or pAd-GFP-p53 (p53) for 24 h and then harvested for extraction of protein or RNA. Insert, detection of p53 expression. (B) HCT-116 (p53−/−) or HCT-116 (+/+) were treated with Doxo at 0.5 or 1.0 μg/ml for 16 h. The cells were separately harvested for protein or RNA. p21 was used as a positive control. (C) MCF-7 cells were transiently transfected with vector (pCMV) or mutant p53 (R175H) and incubated for 24 h. Insert, detection of mutant p53. Values in A, B, and C are means ± SE of 3 separate experiments and normalized to their respective controls as 1. **, P < 0.01.

Fig. 3.

p53 induces the miR-145 promoter activity through binding to p53RE-2. (A) A schematic description of the putative miR-145 promoter with 2 potential p53 response elements, p53RE-1, and p53RE-2 and the mutated p53RE-2 (p53RE-2-mut), as compared with the p53RE consensus, where R = A+G; W = A+T and Y = C+T. Small letters denote deviations from the consensus. The conserved nucleotides C and G are highlighted in red. The sequences between the 2 half sites are indicated with blue color. In p53RE-2-mut, the conserved C and G were replaced by T and A, respectively. (B) and C, Luciferase assays with pMIR-145p-Luc-1 in NIH/3T3 and MCF-7 cells, respectively. pMIR-205p-Luc was used as a negative control. (v) vector. (D) Deletion and site-directed mutagenesis analysis identifies the importance of miR-145 p53RE-2 in p53-mediated induction of the luciferase activity. (E) ChIP assay reveals that p53 specifically interacts with p53RE-2. p21 serves as a positive control. Sequences from the upstream region of the human calcium-activated chloride channel protein 2 gene without p53 binding sites serves as a negative control. 145-p, mir-145 promoter; p21-p, p21 promoter. Values in B–D are means ± SE of 3 separate experiments and normalized to respective controls as 1 (B and C) or 100% (D). **, P < 0.01.

Fig. 4.

miR-145 directly targets c-Myc and inhibits its expression. (A) Western blot shows suppression of the c-Myc protein by miR-145, but not the mutant miR-145 (miR-145-mt). Cells were transfected with vector alone or miR-145 or miR-145-mt for 24 h before harvesting for protein extraction. (B) Blockade of miR-145 by anti-miR145 (Anti-145) causes up-regulation of c-Myc. HCT-116 cells were transfected with scrambled oligo or anti-miR-145 oligo and were harvested 24 h later for protein or RNA extraction. The same transfected cells were separately used for Western blot or TaqMan real-time PCR. (C) Suppression of the c-Myc downstream target genes eIF4E and CDK4 by miR-145 in HCT-116 cells. (D) Effect of miR-145 on cell cycle. HCT-116 cells were transiently transfected with vector control or miR-145 expression vector. Two days later, cells were harvested for cell cycle analysis. (E) A putative miR-145 binding site in the c-Myc 3′-UTR and mutant miR-145 in which the seed sequences were mutated. (F) Effect of miR-145 or mutant miR-145 on the luciferase activity of Luc-c-Myc-UTR and Luc-c-Myc-UTR-d in which the putative miR-145 binding site was deleted. For luciferase assays, 293T cells were transfected with vector or miR-145 expression vector and then harvested for lysis of cells 2 days after transfection. Values in F are means ± SE of 3 separate experiments and normalized to their respective controls as 100%. **, P < 0.01.

Fig. 5.

Role of miR-145 in p53-mediated repression of c-Myc. (A) Effect of wild-type p53 and mutant p53 (R175H) and Doxo-induced p53 on c-Myc expression. HCT-116 cells were transduced with p53 or transfected with mutant p53 for 24 h and then harvested for extraction of protein. For induction of the endogenous p53, the experiment was carried out same as in Fig. 2B with 1 μg/ml Doxo. (B) HCT-116 (p53−/−) and HCT-116 (p53 +/+) were treated with Doxo at 0.5 or 1.0 μg/ml for 16 h. Over 50% repression was observed in the Doxo treated HCT-116 (p53+/+) cell. (C) Blockade of p53-mediated c-Myc repression by anti-miR-145. HCT-116 (p53−/−) and HCT-116 (p53 +/+) cells were first transfected with anti-miR-145 or control oligos. After 10 h, the cells were treated with Doxo at 1.0 μg/ml for 16 h before extraction of protein or RNA. Anti-miR-21 serves as an additional negative control. Blank without Doxo serves as a baseline of c-Myc for comparison between Doxo-treated and Doxo-untreated, or between anti-miR-145 and negative control (scrambled oligo or anti-miR-21). The mock control was performed by the same transfection experiments without oligos to confirm that the repression of c-Myc by Doxo/p53 is specific. Bottom: Relative c-Myc levels are averages ± SE of 3 separate experiments and normalized to the no Doxo control as 100%. The value for the anti-miR-145 lane in HCT-116 (p53 +/+) cells is 92%. **, P < 0.01 compared with blank controls. (D) Effect of c-Myc-siRNA and/or anti-miR-145 on c-Myc, p21 and Bax. HCT-116 cells transfected with scrambled oligo or c-Myc-siRNA for 24 h, followed by the treatment with or without Doxo (0.5 μg/ml) for 16 h. The c-Myc protein blot in the Right was exposed longer than the left 1 to detect various amounts of the c-Myc protein in different treatments. (E) miR-145 also suppresses c-Myc in p53−/− cells.