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Negative regulation of the tumor suppressor p53 gene by microRNAs.

Kumar M, Lu Z, Takwi AA, Chen W, Callander NS, Ramos KS, Young KH, Li Y - Oncogene (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, School of Medicine, University of Louisville, Louisville, KY, USA.

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.

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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.
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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.

Mentions: All values in Fig. 1C, 1D, 1E, 1G, 1H, 2C, 3C, 4B, 4C, 5B, 5D, and 6C were represented as means with SEM from three to six independent experiments. Statistical analyses were performed using Student’s t-test with results considered statistically significant at P≤ 0.05. Pearson Correlation analyses in Fig. 6A were performed using SPSS 11.5 (SPSS, Inc., Chicago, IL). Cell culture, Western blotting, qRT-PCR, cell cycle analysis, apoptosis, and senescence assays were performed using routine procedures and details were provided in Supplemental Text 1.


Negative regulation of the tumor suppressor p53 gene by microRNAs.

Kumar M, Lu Z, Takwi AA, Chen W, Callander NS, Ramos KS, Young KH, Li Y - Oncogene (2010)

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.
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Related In: Results  -  Collection

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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.
Mentions: All values in Fig. 1C, 1D, 1E, 1G, 1H, 2C, 3C, 4B, 4C, 5B, 5D, and 6C were represented as means with SEM from three to six independent experiments. Statistical analyses were performed using Student’s t-test with results considered statistically significant at P≤ 0.05. Pearson Correlation analyses in Fig. 6A were performed using SPSS 11.5 (SPSS, Inc., Chicago, IL). Cell culture, Western blotting, qRT-PCR, cell cycle analysis, apoptosis, and senescence assays were performed using routine procedures and details were provided in Supplemental Text 1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, School of Medicine, University of Louisville, Louisville, KY, USA.

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.

Show MeSH
Related in: MedlinePlus