MicroRNA 125a and its regulation of the p53 tumor suppressor gene - PubMed (original) (raw)

MicroRNA 125a and its regulation of the p53 tumor suppressor gene

Yingjie Zhang et al. FEBS Lett. 2009.

Abstract

MicroRNA (miRNA) are a class of non-coding RNA that suppress gene expression by degradation or translational inhibition of target RNA. Several miRNA have been shown to target oncogenes and recently miRNA-125b was shown to translationally and transcriptionally inhibit the p53 gene. Here, we show that an additional isomer of miRNA-125 (miRNA-125a) translationally arrests mRNA of the p53 tumor suppressor gene. The basis of this activity is the high degree of sequence homology between the seed sequence of miR-125a and the 3'-UTR of p53. Our findings add miRNA-125a to the growing list of miRNA with oncogenic targets.

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Figures

Fig. 1

Fig. 1

miR-125 targets the 3′-UTRof p53. (a) The p53-miRNA-125a/b interactome: both isomers of miR-125 (125a, 125b) share homology in their seed sequence (5′ nt 2–7) with the 3′-UTR of p53. (b) Ectopic miRNA-125a expression vector. HEK 293T cells were transfected with miRNA-125a expression vector or control vectors that harbored either irrelevant DNA construct encoding miRNA-378 or no insert (EV). Levels of mature miRNA-125a were visualized by Northern blot (left panel) and quantified by Real Time PCR (right panel). Both assays revealed that wild-type HEK 293T cells express low levels of miRNA-125a that were increased by ∼12-fold upon transfection of miRNA-125a as quantified by Real Time PCR in three independent experiments (results are reported as mean values ±S.D.). (c) Regulation of the p53 3′-UTR by miRNA-125a. Activity of miRNA-125a on the 3′-UTRof p53 was initially assessed by luciferase based reporter assays. The p53 3′-UTR was incorporated into the firefly luciferase gene and run off a single promoter. A constitutively expressed Renilla gene served to normalize transfections. All constructs were introduced into HEK 293T cells with miRNA-125a or an empty control vector (EV) and luminescence was measured at 48 h. miRNA-125a reduced luciferase levels by 40%. The sequence specificity of miRNA-125a/p53 3′-UTR interaction was probed by mutating the seed sequence of miRNA-125a in positions 2 and 4. In comparison to wild-type miRNA-125a, the mutated version had negligible effect (<5%) on the p53 3′-UTR as quantified by levels of luciferase reduction after transfection. Data are presented as mean fold reduction ± S.D. All experiments were performed in triplicate.

Fig. 2

Fig. 2

miRNA-125a reduces p53 protein levels by translational arrest of p53 mRNA. (a) Ectopic expression of miRNA-125a reduces p53 protein expression in HEK 293T cells by translational arrest. Cells were transfected with expression vectors for miRNA-125 or an irrelevant construct encoding miRNA-378 and p53 protein levels were quantified by Western blot. GAPDH levels were used to normalize protein input (upper panel). p53 protein reduction by miRNA-125a was not associated with reduction in p53 mRNA levels as quantified by Real Time PCR in cells that had been treated with an empty vector or one encoding miRNA-125a. Data are presented as mean fold reduction ± S.D. All experiments were performed in triplicate. (b) Efficacy of miRNA-125a approaches that of p53 short interfering RNA (siRNA). p53 protein levels were quantified by Western blot in a breast cancer cell line (MCF-7) that had been transfected with either p53 specific siRNA or miRNA-125a. Both agents reduced protein levels of both p53 and downstream protein p21. GAPDH levels were used to normalize protein input. (c) The effect of p53 levels themselves on miRNA-125a was examined in three cell lines. Reduction of p53 protein levels by siRNA had no effect upon levels of endogenously expressed miRNA-125a (upper panel) or miRNA-125b (lower panel) when compared to cells that had been treated with a control empty vector (EV). (d) Several proteins including COP1 and Prih2 are known to negatively regulate p53 protein levels. We found that ectopic expression of miRNA-125a had no effect on levels of either protein, as assessed by Western blot.

Fig. 3

Fig. 3

p53-specific genotypic and phenotypic sequelae of miR-125a expression. (a) p53-specific genotypic sequelae of miRNA-125a expression. miRNA-34a/b/c have been established as transcriptional targets of p53. Levels of all three miRNAs were reduced by 20–80% in cells over expressing miRNA-125a as quantified by Real Time PCR of mature (upper panel) and pri-miRNA levels (lower panel). Data are presented as mean fold reduction ± S.D. compared to cells treated with an empty vector (EV). All experiments were performed in triplicate. (b) p53-specific phenotypic sequelae of miRNA-125a expression. Over-expression of miRNA-125a protects cells from cell death induced by arsenic trioxide. Cells were treated with arsenic trioxide and their viability was quantified by MTT assay. As expected, EV treated cells could not withstand the toxicity of arsenic trioxide. Pre-treatment of cells with siRNA targeting p53 or miRNA-125a preserved viability. All experiments were performed in triplicate and the average and standard deviation of viable cells is shown. (c) The response to arsenic trioxide was assessed by BrdU assay in cells with p53 knockdown or in cells treated with an irrelevant empty vector or one encoding miRNA-125a. Arsenic trioxide or its absence was not related to any significant differences in cellular proliferative capacity in any of the six conditions tested.

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