Abstract
miR-34, a tumor suppressor miRNA family transcriptionally activated by p53, is considered a critical mediator of p53 function. However, knockout of the mouse miR-34 family has little or no effect on the p53 response. The relative contribution of different miR-34 family members to p53 function or how much p53 relies on miR-34 in human cells is unclear. Here we show that miR-34a has a complex effect on the p53 response in human cells. In HCT116 cells miR-34a overexpression enhances p53 transcriptional activity, but the closely related family members, miR-34b and miR-34c, even when over-expressed, have little effect. Both TP53 itself and MDM4, a strong p53 transactivation inhibitor, are direct targets of miR-34a. The genes regulated by miR-34a also include four other post-translational inhibitors of p53. miR-34a overexpression leads to variable effects on p53 levels in p53-sufficient human cancer cell lines. In HCT116, miR-34a overexpression increases p53 protein levels and stability. About a quarter of all mRNAs that participate in the human p53 network bind to biotinylated miR-34a, suggesting that many are direct miR-34a targets. However, only about a fifth of the mRNAs that bind to miR-34a also bind to miR-34b or miR-34c. Two human cell lines knocked out for miR-34a have unimpaired p53-mediated responses to genotoxic stress, like mouse cells. The complex positive and negative effects of miR-34 on the p53 network suggest that rather than simply promoting the p53 response, miR-34a might act at a systems level to stabilize the robustness of the p53 response to genotoxic stress.
Highlights
Genotoxic stress activates p53 transcription of many genes, leading to cell cycle arrest, apoptosis or senescence [1]
To assess the effect of miR-34a on the p53 response, we used qRT-PCR to analyze the effect of miR-34a overexpression on 9 p53-activated gene mRNAs in wild type (WT) and TP53-/HCT116 cells. miR-34a overexpression increased all but one of the targets tested (BAX, MDM2, PUMA, PIG3, FAS, CDKN1A, GADD45A and TP53INP1, but not PMAIP1 (NOXA)), but only in WT cells (Fig 1A)
We used luciferase reporter promoter assays, in p53-sufficient HCT116 cells, to assess whether miR-34 overexpression enhanced promoter activities of a sequence of 13 tandem repeats of the p53 binding site [16] or the promoters of p53-regulated genes, PUMA, CDKN1A and BAX. miR-34a overexpression increased by 4-fold the luciferase activity of the pG13-luc, CDKN1A and PUMA promoters and increased by 2-fold BAX promoter activity (Fig 1C). miR-34b-5p overexpression had a modest, but significant, effect on 2 of the 4 promoters, while miR-34c did not significantly increase activity of any (Fig 1C), even though it was overexpressed more than a hundred fold above its endogenous level after genotoxic stress
Summary
Genotoxic stress activates p53 transcription of many genes, leading to cell cycle arrest, apoptosis or senescence [1]. Multiple miRNAs, including the miR-34 family, are transcriptionally activated by p53. In turn miRNAs regulate expression of many p53-induced genes [2, 3]. The miR-34 family consists of 3 miRNAs—miR-34a on human chromosome 1p36 and miR-34b/c, co-transcribed on human chromosome 11q23. MiR-34a is expressed in most tissues, while miR-34b/c are predominantly expressed in lung and testis [4, 5]. Three chromosome 5q11.2 miRNAs (miR-449a/b/c) share a seed sequence with miR-34, and have a tissue
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