Negative regulation of the tumor suppressor p53 gene by microRNAs - PubMed (original) (raw)
Negative regulation of the tumor suppressor p53 gene by microRNAs
M Kumar et al. Oncogene. 2011.
Abstract
The tumor suppressor p53, encoded by the TP53 gene, is recognized as the guardian of the human genome because it regulates many downstream genes to exercise its function in cell cycle and cell death. Recent studies have revealed that several microRNAs (miRNAs) are important components of the p53 tumor suppressor network with miR-125b and miR-504 directly targeting TP53. In this study, we use a screening method to identify that two miRNAs (miR-25 and miR-30d) directly target the 3'UTR of TP53 to downregulate p53 protein levels and reduce the expression of genes that are transcriptionally activated by p53. Correspondingly, both miR-25 and miR-30d adversely affect apoptotic cell death, cell cycle arrest and cellular senescence. Inhibition of either miR-25 or miR-30d expression increases endogenous p53 expression and elevates cellular apoptosis in several cell lines, including one from multiple myeloma that has little TP53 mutations. Thus, beyond miR-125b and miR-504, the human TP53 gene is negatively regulated by two more miRNAs: miR-25 and miR-30d.
Conflict of interest statement
Disclosure of Conflicts of Interest
The authors declare no conflicts of interest.
Figures
Figure 1
A screening assay to identify miRNAs targeting TP53 in 293T cells. (A) Schematic presentation of the tier-1 assay measuring the basal p53 activity. (B) Schematic presentation of the tier-2 assay. (C) The tier-1 assay for 8 miRNAs that are predicted to target TP53. (D) The tier-2 assay for 8 miRNAs that are predicted to target TP53. (E) The protein levels of p53 were down-regulated by miR-25 and miR-30d. “p53 RQ” indicated the relative quantification of p53 normalized to β-actin. (F) TP53 3’UTR mutations. Three mutations were created in the TP53 3’UTR to disrupt the respective seed sequence: 3’UTR interaction for miR-25, miR-30d, and miR-200a. Red vertical bars denote mutant while black wt. (G) Rluc down-regulation by miRNAs is dependent on the 3’UTR of TP53. 293T cells were cotransfected with pRL-TK carrying a wt or mutant 3’UTR sequence. (H) p53 mRNA levels were decreased with miRNA overexpression. * _P_≤ 0.05 with n=3–6.
Figure 2
miRNAs down-regulate p53 expression and reduce apoptosis and cell cycle arrest in 293T cells. (A) p53, p21, Bax, and Gadd45α were down-regulated in 293T cells transfected with miRNAs with or without etoposide treatment. (B) Caspase 9 activities in 293T cells overexpressing miRNAs with or without etoposide treatment; the Y-axis denotes the relative luminescent units (RLU) from the Caspase-Glo® 9 Assay (n=4). (C) Caspase 3,7 activities in 293T cells overexpressing miRNAs with or without etoposide treatment; the Y-axis denotes the relative fluorescent units (RFU) from the Apo-ONE® Homogeneous Caspase-3/7 Assay (n=4). (D) miRNAs inhibit p53-mediated G2 arrest in cells treated with etoposide; the X axis denotes events (the number of cells) and Y denotes the emitted fluorescent light (DNA content) of the DNA dye (PI) (n=6). *, _P_≤ 0.05 and **, _P_≤ 0.01.
Figure 3
miRNAs down-regulate p53 expression and reduce apoptosis and cell cycle arrest in H1299 cells. (A) p53, p21, Bax, and Puma were down-regulated in H1299 cells transfected with miRNAs and an ectopic p53 with a wt 3’UTR, but not those with mutant 3’UTRs. (B) A representative photo of flow-cytometry used to determine cellular apoptosis of H1299 cells. The Y-axis denotes the log values of signal density for Annexin V with X to that of PI. The percentage of cells in three quadrants was presented. (C) The bar graph for 6 independent runs of (B). Student’s t-tests were performed for samples with or without etoposide treatment, respectively. (D) miRNAs inhibit p53-mediated G1 arrest. “Control” was performed using parental vectors only (1:1 of miRNA empty vector and p53 empty vector). The X axe denotes events (the number of cells) and Y denotes the emitted fluorescent light of the DNA dye (PI), i.e., DNA content. * _P_≤ 0.05 with n=3–6.
Figure 4
miRNAs inhibit p53 cellular senescence expression in HCT116 and HCT116 p53−/− cells. (A) p53, p21, and Bax expression in HCT116 and HCT116 p53−/− cells with miRNA overexpression. (B) miRNAs inhibit cellular senescence in HCT116 cells. (C) miRNAs inhibit cellular senescence in HCT116 p53−/− cells expressing an ectopic wt p53 with a wt 3’UTR, but not those with mutant 3’UTRs. The bar graphs in (B) and (C) represent 3 independent runs. “Control” in (C) was performed using parental vectors only (1:1 of miRNA empty vector and p53 empty vector). The Y axis in the graphs denotes the number of senescence-positive cells per microscope field. * _P_≤ 0.05 with n=6.
Figure 5
Down-regulation of miRNAs increases p53 expression and cellular apoptosis in HCT116 and A549 cells. (A) Inhibition of miRNA expression using miRNA inhibitors upregulates the expression of p53 and its target genes in HCT116 cells. (B) Inhibition of miRNA expression promotes cellular apoptosis in HCT116 cells. (C) and (D), similar experiments but in lung cancer A549 cells. “Neg contl”, the negative control; “Anti-miR-25/-30d”, the inhibitors to miR-25/-30d. * _P_≤ 0.05 with n=6.
Figure 6
Down-regulation of miRNAs increases p53 expression and cellular apoptosis in multiple myeloma cells. (A) miR-25, miR-30d, and TP53 mRNA expression in plasma cells from bone marrow aspirates of multiple myeloma patients (MM) and healthy donors (NC). Whisker-box plot where the boxes indicate the 25th and 75th percentile; thin lines in the boxes indicate the 50th percentile and thick lines denote the mean values; whisker caps indicate the 5th and the 95th percentile; filled circles indicate outliers. (B) Inhibition of miRNA expression using miRNA inhibitors increases the expression levels of p53 and its target genes in H929 cells. (C) Inhibition of miRNA expression promotes cellular apoptosis in H929 cells. “Neg contl”, the negative control; “Anti-miR-25/-30d”, the inhibitors to miR-25/-30d. * _P_≤ 0.05 with n=6.
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