Several microRNAs mediate the functions of p53 family members. Here we characterize miR‐1246 as a new target of this family. In response to DNA damage, p53 induces the expression of miR‐1246 which, in turn, reduces the level of DYRK1A, a Down syndrome‐associated protein kinase. Knockdown of p53 has the opposite effect. Overexpression of miR‐1246 reduces DYRK1A levels and leads to the nuclear retention of NFATc1, a protein substrate of DYRK1A, and the induction of apoptosis, whereas a miR‐1246‐specific inhibitor prevented the nuclear import of NFATc1. Together, these results indicate that p53 inhibits DYRK1A expression through the induction of miR‐1246.
The p53 tumour suppressor and its homologues, p63 and p73, exert their anti‐tumour functions primarily by their transcriptional activity to induce the expression of protein‐coding genes and microRNAs (miRNAs) responsible for apoptosis, cell growth arrest, differentiation and senescence (He et al, 2007; Raver‐Shapira et al, 2007; Braun et al, 2008; Sachdeva et al, 2009; Sampath et al, 2009; Belyi et al, 2010; Su et al, 2010). However, their roles in animal development are distinct: p63 is more important for epithelial development (Mills et al, 1999); p73 is for the development of the central nervous and immune systems (Yang et al, 2000); and p53 is almost unnecessary for animal development (Donehower et al, 1992). In contrast to the many target genes identified for the p53 homologues (Stiewe, 2007), few miRNAs are known to be their targets (He et al, 2007), particularly specific to each of the homologues (Sampath et al, 2009; Su et al, 2010). In our initial attempt to identify possible miRNAs as targets for each homologue, we identified miR‐1246 as a new transcriptional target for the p53 family. This study shows a new p53 regulatory pathway, involving miR‐1246, DYRK1A and NFAT, that is potentially important for tumorigenesis, suggesting that the p53 family might have a regulatory role in Down syndrome development.
Results And Discussion
Identification of miR‐1246 as a new p63 target
To identify p63‐specific miRNA targets, we conducted an miRNA array analysis of RNA samples isolated from TAp63γ or ΔN‐p63γ (also known as p40) expressed human lung carcinoma H1299 cells (Fig 1A), and found miR‐1246 to be the most likely candidate target of TAp63γ (Fig 1B; supplementary Fig S1 online). Interestingly, miR‐1246 was only found in the human and ape genome, and its human gene is found on chromosome 2q31.1 (supplementary Fig S2A online). This result was confirmed by using quantitative reverse transcription (qRT)–PCR assays, as miR‐1246 was markedly induced by TAp63γ (Fig 1C), but not ΔN‐p63γ in H1299 cells (data not shown). Conversely, short‐interfering RNA (siRNA) knockdown of endogenous p63 in human Hacat keratinocytes reduced the miR‐1246 level (Fig 1D). Although TAp63γ and ΔN‐p63α were knocked down by this siRNA (Fig 1D), the decrease of miR‐1246 levels is probably due to the reduction of TAp63γ, but not ΔN‐p63α, because overexpression of the latter failed to induce this miRNA (Fig 1A–C and data not shown). The genomic sequence encoding this miRNA has a highly conserved p53‐responsive DNA element (p53RE) −194 bp upstream from the transcriptional initiation site (Fig 2A). To test if p63 binds to this p53RE sequence, we conducted a chromatin‐associated immunoprecipitation (ChIP) assay after treating Hacat cells with etoposide using primers for the promoter or a p53RE‐deficient DNA sequence. Endogenous TAp63γ specifically bound to this miR‐1246 promoter, but not to the control DNA sequence, as pulled down with 4A4 antibodies, but not nonspecific immunoglobulin G (Fig 1E). As shown in supplementary Fig S3B online, the ectopic p63 also bound to the miR‐1246 promoter, but not to a control DNA location. Furthermore, we assessed luciferase expression driven by either a wild‐type or a mutant‐p53RE‐motif‐containing miR‐1246 promoter (Fig 2D) in H1299 cells. TAp63γ, but not ΔN‐p63γ, markedly induced luciferase activity when wild‐type miR‐1246 promoter was used (Fig 2E; supplementary Fig S3A online). These results show that p63 induces the transcription of miR‐1246.
miR‐1246 is a target for the p53 family
To determine whether miR‐1246 is a target for the p53 family, we overexpressed either p53 or p73 in H1299 cells and found that they can both induce the expression of this miRNA and p21 (Fig 2B), another p53 target (el‐Deiry et al, 1993). In response to DNA damage caused by etoposide, miR‐1246, similarly to p21, was induced in wild‐type, but not mutant or null p53‐containing human ovarian cancer cell lines (Fig 2C). This result was reproduced in other wild‐type p53‐containing human cancer cell lines (data not shown). In addition, knocking down p53 abrogated the induction of miR‐1246 expression in response to DNA damage (supplementary Fig S4B online). Similarly, both p53 and TAp73β induced the luciferase expression driven by the wild‐type miR‐1246 promoter (Fig 2E). Finally, p53 bound to the endogenous miR‐1246 promoter, measured by ChIP assay (Fig 2F; supplementary Fig S4C online). These results indicate that miR‐1246 is a new transcriptional target of the p53 family . Next, we focused on the functional effect of p53 on this miRNA.
miR‐1246 targets DYRK1A on p53 activation
By using bioinformatic analysis using experimentally derived rules for miRNA target recognition (Bartel, 2009), we identified some target messenger RNAs (mRNAs) of miR‐1246. One candidate mRNA with an ideal miR‐1246‐targeted 3′‐untranslated region (3′UTR) sequence encodes DYRK1A (Fig 3A), a dual‐specificity tyrosine (Y) phosphorylation‐regulated kinase (Becker et al, 1998) that is highly expressed in patients with Down syndrome due to trisomy 21 (Kline et al, 2000). Overexpression of miR‐1246, but not of control miRNA, in H1299 cells using the pSIR–miRNA vector reduced DYRK1A levels, as indicated by western blot analysis (Fig 3B). We then co‐transfected p53‐null human colon cancer HCT116 cells with the pSIF–miR‐1246 expression vector and a luciferase reporter plasmid that carried either a wild‐type or a mutant 3′UTR sequence derived from the DYRK1A mRNA (Fig 3C), and measured luciferase activity. MiR‐1246, but no control miRNA, reduced the luciferase activity when only the wild‐type 3′UTR sequence‐luciferase reporter was used (Fig 3D). These results indicate that miR‐1246 targets the 3′UTR sequence of the DYRK1A mRNA, reducing DYRK1A protein levels.
Consistent with the above results, siRNA knockdown of p53 in U2OS and HCT116 cells reduced the level of endogenous miR‐1246, but increased the level of DYRK1A (Fig 3E; supplementary Fig S4A online). Etoposide induced p53 and miR‐1246 (Figs 2C and 3F), but reduced DYRK1A levels (Fig 3F) and luciferase activity from a reporter plasmid with the wild‐type, but not the mutant, miR‐1246‐targeted 3′UTR sequence of the DYRK1A mRNA (Fig 3G). The levels of p53 and miR‐1246 were inversely proportional to those of DYRK1A in several human cancerous cells, lines tested (supplementary Fig S6 online), except MCF7 cells, in which p53 was less active due to highly expressed MDM2 and MDMX (Midgley & Lane, 1997). Next, we introduced either an inhibitor specific to this miRNA or a control inhibitor into p53‐containing human lung carcinoma A549 cells, and then treated the cells with etoposide followed by western blot analysis and qRT–PCR. Again, etoposide treatment induced p53 and reduced the level of DYRK1A (Fig 4A). By contrast, the miR‐1246 inhibitor, but not its control, markedly increased the level of DYRK1A, regardless of the presence of etoposide (Fig 4A). This effect was specific to miR‐1246 (right panel of Fig 4A), but not p21 (left panel of Fig 4A). These results indicate that DNA damage signals induce p53‐dependent expression of miR‐1246, which represses the expression of DYRK1A by targeting its 3′UTR RNA sequence.
miR‐1246 inhibits DYRK1A activity
DYRK1A has been shown to phosphorylate NFATc1 (Arron et al, 2006; Gwack et al, 2006), a transcriptional factor that is essential for immune regulation and organ development (Shaw et al, 1988; Xanthoudakis et al, 1996) and linked to Down syndrome and cancer (Ryeom et al, 2008; Baek et al, 2009; Wu et al, 2010), and to suppress its functions by promoting its nuclear export (Arron et al, 2006; Gwack et al, 2006). MiR‐199b, a transcriptional target of NFAT, was shown to target DYRK1A, thus forming a negative feedback loop between NFAT and DYRK1A (da Costa Martins et al, 2010). We aimed to determine whether miR‐1246 affects this activity of DYRK1A by establishing a stable green fluorescent protein (GFP)–NFATc1 expressing U2OS cell line (Arron et al, 2006) and transfecting the cells with miR‐1246 mimic, its control mimic, the miR‐1246 inhibitor or its negative control inhibitor, as shown in Fig 4A. Cells were then treated with 1 μM thapsigargin to deplete cellular Ca2+ by inhibiting the sarcoplasmic/endoplasmic reticulum ATPase and consequently activate the calmodulin‐dependent phosphatase calcineurin, which in turn dephosphorylates NFATc1 and promotes its nuclear import (Beals et al, 1997). After 60 min, cells were treated with 20 μg/ml cyclosporin A (CsA), which inactivates calcineurin and thus reverses the phosphorylation status of NFATc1, endorsing its nuclear export (Nair et al, 1994). Transfected live cells were observed, their images were taken under a fluorescence microscope and part of the cells was used for western blot analysis. Consistent with previous reports (Beals et al, 1997; Arron et al, 2006), GFP–NFATc1 showed a nuclear import followed by a nuclear export when cells were treated with thapsigargin followed by CsA (Fig 4C). However, introduction into the cells of miR‐1246 mimics that reduced the level of endogenous DYRK1A (Fig 4B), but not its control mimic (Fig 4C), prevented the nuclear export of GFP–NFATc1 (Fig 4C). Conversely, overexpression of the miR‐1246 inhibitor, but not its negative control (Fig 4C), blocked the nuclear import of NFATc1 by reducing miR‐1246 and increasing DYRK1A levels (Fig 4A). In miR‐1246 mimics and inhibitors, more than 80% of GFP–NFATc1, expressing cells showed nuclear retention or exclusion of NFATc1, as shown in Fig 4C. Additionally, overexpression of miR‐1246 enhanced the NFATc1 transcriptional activity (Fig 4D) to produce interleukin‐2 (supplementary Fig S5 online), whereas the miR‐1246 inhibitor suppressed this activity (Fig 4D). Moreover, as DYRK1A was shown to suppress apoptosis (Laguna et al, 2008), overexpression of miR‐1246 mimic, but not its control mimic, dramatically induced apoptosis, indicated by the increase of sub‐G1 cells (Fig 4E). These results indicate that miR‐1246 inactivates DYRK1A by repressing its expression.
In summary, we identify miR‐1246 as a new transcriptional target for the p53 family (Figs 1,2). This miRNA downregulates the expression of DYRK1A by targeting its 3′UTR mRNA sequence, consequently activating NFAT1c and inducing apoptosis (Figs 3,4). Our study shows that p53 can repress the expression of DYRK1A through induction of miR‐1246 and identifies a new downstream effector for this tumour suppressor (Fig 4F). Similarly, p63 and p73 also induced the expression of miR‐1246 (Figs 1,2; supplementary Fig S3C online). Previous studies showed that DYRK1A can inactivate NFATc1 by phosphorylating it and excluding it from the nucleus (Arron et al, 2006; Gwack et al, 2006), whereas NFATc1 was recently shown to be required for the anti‐cancer function of p53 in an animal model (Wu et al, 2010) and to be downregulated in skin cancers (Wu et al, 2010). Moreover, DYRK1A was shown to have anti‐apoptotic activity (Laguna et al, 2008). Predictably, suppression of either NFATc1 activity or apoptosis by DYRK1A impairs p53 function. Hence, it is logical that p53 uses miR‐1246 to deactivate DYRK1A (Fig 4F).
As DYRK1A is a key causative factor in the development of Down syndrome (Guimera et al, 1996; Song et al, 1996), our study not only identifies a possible new role of the p53 family in Down syndrome development through miR‐1246, but also offers a native and useful molecule for the development of a therapeutic anti‐Down‐syndrome agent, which can be tested in a DYRK1A transgenic mouse model (Fernandez et al, 2007), as the miR‐1246‐targeted 3′UTR sequences derived from human and mouse DYRK1A mRNAs are identical (supplementary Fig S2B online).
Cell lines. Unless indicated, all cells (H1299, A549, U2Os, HCT116) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 0.1 mg/ml streptomycin at 37 °C in 5% CO2. SKOV3, A2780 and OV90 were obtained from Daniela E. Matei (IUSM) and cultured in DGEM (DMEM/M199=1:1), supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 0.1 mg/ml streptomycin at 37 °C in 5% CO2. Human Hacat keratinocytes were obtained from Dan F. Spandau (Indiana University School of Medicine).
ChIP–PCR. ChIP analysis was performed as described previously (Zeng et al, 2002) using p53 (DO‐1) antibodies for exogenous p53 and Flag antibodies for overexpressed p63. Immunoprecipitated DNA fragments were analysed by semiquantitative and/or real‐time PCR amplification using primers for miR‐1246 and control genes. The primers are listed in supplementary Table S1 online.
Nuclear localization of NFATc1. U2OS cells, stably expressing GFP–NFATc1 (U2OS‐NFATC1), were generated in the laboratory. Cells were transfected with miR‐1246 mimic, inhibitor or negative control, as described as above. 36 hours after transfection, cells were treated with thapsigargin at a final concentration of 1 μM, images were taken under a Zeiss fluorescence microscope at the indicated times. 60 min after thapsigargin treatment, cells were washed twice with medium, followed by addition of CsA (final concentration 20 μg/ml) to the medium. Images of live cells were taken at the indicated time points. The result in Fig 4C was observed in more than 80% of GFP–NFATc1‐expression cells.
See the supplementary information online for plasmids, adenovirus, antibodies, chemicals, RT–PCR, real‐time qPCR, transient transfection, immunoprecipitation, western blot, knockdown of endogenous miRNAs and p53, flow cytometry and luciferase reporter assays.
Supplementary information is available at EMBO reports online (http://www.emboreports.org).
Conflict of Interest
The authors declare that they have no conflict of interest.
We thank G.R. Crabtree, P.J. Roach, A.V. Skurat, R. Wek, D.E. Matei, D.F. Spandau, and Y. Li for cell lines and reagents. H.L. was also supported by National Institutes of Health‐National Cancer Institute grants CA127724, CA095441 and CA129828.
Author contributions: Y.Z, J.M.L. and H.L. designed the experiments; Y.Z conducted most of the studies; J.M.L. conducted part of the ChIP, real‐PCR analyses, and immunofluorescence experiments; S.X.Z. conducted miRNA identification and part of the immunofluorescence experiments; Y.Z., J.M.L. and H.L. analysed the data and composed the manuscript; H.L drafted the manuscript.
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