1. Negative Regulation of Tumor Suppressor p53 by MicroRNA miR-504
Wenwei Hu, Chang S. Chan, Rui Wu, Cen Zhang, Yvonne Sun, Jun S. Song, Laura H. Tang, Arnold J. Levine, Zhaohui Feng 
Molecular Cell 
Volume 38, Issue 5, Pages (June 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1 miR-504 Negatively Regulates p53 Protein Levels in Cells
(A) p53 protein levels in cells transfected with different miRNA oligonucleotides. Human HCT116 p53+/+, H460, and MCF7 cells were transfected with various miRNA oligonucleotides (100 nM), and the p53 protein levels were measured at 24 hr after transfection by Western blot assays. Control (con) cells were transfected with scrambled miRNA oligonucleotides.
(B) Relative levels of the p53 protein in human cells transfected with miR-504 oligonucleotides. The p53 protein levels in (A) were normalized with actin. The expression levels of p53 in control cells transfected with scrambled miRNA oligonucleotides were designated as 1. Data are presented as mean ± SD (n = 3).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 2
miR-504 Binding Sites in Human p53 3′UTR Mediates the Downregulation of p53 Protein Expression by miR-504
(A) miR-504 sequences and its putative binding sites in the 3′UTR of human p53. The drawing is not to scale.
(B) Luciferase reporter constructs containing wild-type or mutant human p53 3′UTR fused at the 3′ end of the firefly luciferase gene. Shown are luciferase reporter vectors with wild-type human p53 3′UTR (1), with mutant deletion of putative binding site 1 (2), site 2 (3), or both sites (4) for miR-504.
(C) The miR-504 binding sites in human p53 3′UTR mediates the repression of luciferase activities in cells. HCT116 p53+/+ were transfected with four different luciferase constructs described in (B) together with miR-504 oligonucleotides (100 nM) or the scrambled miRNA oligonucleotides as a control (con). Luciferase activities were measured at 24 hr after transfection. Data are presented as mean ± SD (n = 3).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 3
miR-504 Decreases p53 Protein Accumulation and Transcriptional Activity in Response to Stress
(A) Overexpression of miR-504 downregulates protein levels of p53 and p53 target genes, MDM2 and p21, in HCT116 p53+/+ treated with Etoposide. HCT116 p53+/+ and p53−/− cells were transfected with 100 nM miR-504 oligonucleotides or scrambled miRNA oligonucleotides (con). At 24 hr after transfection, cells were treated with Etoposide (Etp, 20 μM). Protein levels were analyzed at 8 hr after Etoposide treatment.
(B) The mRNA expression patterns of p53 target genes in HCT116 cells with miR-504 overexpression. HCT116 p53+/+ and p53−/− cells were transfected with miR-504 oligonucleotides or control oligonucleotides (con). Cells were treated with Etoposide (Etp, 20 μM) at 24 hr after transfection. The mRNA expression of p53 target genes was analyzed by real-time PCR at 8 hr after Etoposide treatment. The expression of all genes was normalized to Actin. The gene expression levels in control cells (con) transfected with scrambled oligonucleotides were designated as 1. Data are presented as mean ± SD (n = 3).
(C) miR-504 expression downregulates protein levels of p53, MDM2, and p21 in HCT116 p53+/+ treated with IR. Cells were transfected with miR-504 oligonucleotides, then treated with IR (10 Gy) at 24 hr after transfection. Protein levels were analyzed at 8 hr after IR.
(D) miR-504 downregulates p53 mRNA levels in cells. MCF7, H460, and HCT116 p53+/+ cells were transfected with miR-504 oligonucleotides for 24 hr before p53 mRNA levels were measured by real-time PCR. The levels of the p53 mRNA in control cells were designated as 1. Data are presented as mean ± SD (n = 3).
(E) Stable ectopic miR-504 expression downregulates protein levels of p53, MDM2, and p21 in cells. HCT116 p53+/+ cells with stable miR-504 overexpression by miR-504 expression vectors were treated with Etoposide (Etp, 20 μM), and protein levels were analyzed at 8 hr after treatment. Control cells were stably transfected with control empty vectors.
(F) Inhibition of endogenous miR-504 increases p53 protein levels and transcriptional activity. Human A498 cells were transfected with anti-miR-504 oligonucleotides (100 nM) 24 hr before Etoposide treatment (Etp, 20 μM). Protein levels were measured at 8 hr after Etoposide treatment.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 miR-504 Reduces p53-Mediated Apoptosis
(A) miR-504 reduces p53-mediated apoptosis in U2OS cells. U2OS cells were transfected with 100 nM miR-504 oligonucleotides (miR-504) or scrambled miRNA oligonucleotides (miR-con). Cells were treated with 20 μM Etoposide (Etp) at 24 hr after transfection, and apoptosis were measured at 24 and 36 hr after Etoposide treatment. To show the p53-dependent apoptosis induced by Etoposide, cells were transfected with p53 siRNA oligonucleotides (si-p53; 100 nM) or cotransfected with miR-504 oligonucleotides and p53 siRNA (miR-504+si-p53) at 24 hr before Etoposide treatment. For controls, cells were either transfected with control siRNA oligonucleotides (si-con) or control miRNA together with control siRNA (miR-con+si-con). Data are presented as mean ± SD (n = 3). ∗: p < 0.05 (treated versus control).
(B) miR-504 reduces p53-mediated apoptosis in H460 cells. Cells were treated and apoptosis was measured as described in Figure 4A. Data are presented as mean ± SD (n = 3). ∗: p < (treated versus control).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

6. Figure 5 miR-504 Reduces p53-Mediated Cell-Cycle Arrest
(A) HCT116 p53+/+ (left panel) and p53−/− cells (right panel) were transfected with miR-504 oligonucleotides (miR-504; 100 nM) or scrambled miRNA oligonucleotides (con). At 24 hr after transfection, cells were treated with Etoposide (Etp, 20 μM) and cell-cycle arrest was analyzed in a flow cytometry at 36 hr after Etoposide treatment. Data are presented as mean ± SD (n = 3). p < 0.01 (G1 in #2 versus #4); p < 0.01 (G1 in #2 versus #6).
(B) HCT116 p53+/+ cells with stable overexpression of miR-504 by miR-504 expression vectors (p53+/+-miR-504) and cells stably transfected with a control empty vector (p53+/+-Con) were treated with Etoposide (Etp, 20 μM), and cell-cycle arrest was analyzed at 36 hr after Etoposide treatment. Data are presented as mean ± SD (n = 3). p < 0.01 (G1 in #2 versus #4).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 miR-504 Promotes Tumorigenicity of Cells In Vivo
(A) Nude mice were inoculated (s.c.) with HCT116 p53+/+ or HCT116 p53+/+ cells stably transfected with control empty vectors (p53+/+-control) on the left flank, and were inoculated with HCT116 p53−/− cells or HCT116 p53+/+ cells with stable miR-504 overexpression by miR-504 expression vectors (p53+/+-miR-504) on the right flank, respectively (n = 10 mice/group). Mice at day 21 after inoculation of tumor cells are presented.
(B) Nude mice were inoculated (s.c.) with HCT116 p53+/+ on the left flank and HCT116 p53−/− cells on the right flank, respectively (n = 10 mice/group). Tumor volumes (mm3 = 1/2 [length × width2]) were measured every 3 days after inoculation for 3 weeks. Data are presented as mean ± SD (n = 10).
(C) Nude mice were inoculated with HCT116 p53+/+ cells with stable miR-504 overexpression by miR-504 expression vectors on the right flank (miR-504) and control HCT116 p53+/+ cells stably transfected with empty vectors (con) on the left flank. Data are presented as mean ± SD (n = 10).
(D) Nude mice were inoculated with HCT116 p53−/− cells with stable miR-504 overexpression on the right flank (miR-504) and control HCT116 p53−/− cells stably transfected with empty vectors (con) on the left flank (n = 10 mice/group). Data are presented as mean ± SD (n = 10).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions