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. 2012;7(8):e42034.
doi: 10.1371/journal.pone.0042034. Epub 2012 Aug 1.

MicroRNA-34a modulates MDM4 expression via a target site in the open reading frame

Pooja Mandke  1 , Nicholas Wyatt, Jillian Fraser, Benjamin Bates, Steven J Berberich, Michael P Markey
Affiliations

MicroRNA-34a modulates MDM4 expression via a target site in the open reading frame

Pooja Mandke et al. PLoS One. 2012.

Abstract

Background: MDM4, also called MDMX or HDMX in humans, is an important negative regulator of the p53 tumor suppressor. MDM4 is overexpressed in about 17% of all cancers and more frequently in some types, such as colon cancer or retinoblastoma. MDM4 is known to be post-translationally regulated by MDM2-mediated ubiquitination to decrease its protein levels in response to genotoxic stress, resulting in accumulation and activation of p53. At the transcriptional level, MDM4 gene regulation has been less clearly understood. We have reported that DNA damage triggers loss of MDM4 mRNA and a concurrent increase in p53 activity. These experiments attempt to determine a mechanism for down-regulation of MDM4 mRNA.

Methodology/principal findings: Here we report that MDM4 mRNA is a target of hsa-mir-34a (miR-34a). MDM4 mRNA contains a lengthy 3' untranslated region; however, we find that it is a miR-34a site within the open reading frame (ORF) of exon 11 that is responsible for the repression. Overexpression of miR-34a, but not a mutant miR-34a, is sufficient to decrease MDM4 mRNA levels to an extent identical to those of known miR-34a target genes. Likewise, MDM4 protein levels are decreased by miR-34a overexpression. Inhibition of endogenous miR-34a increased expression of miR-34a target genes and MDM4. A portion of MDM4 exon 11 containing this 8mer-A1 miR-34a site fused to a luciferase reporter gene is sufficient to confer responsiveness, being inhibited by additional expression of exogenous mir-34a and activated by inhibition of miR-34a.

Conclusions/significance: These data establish a mechanism for the observed DNA damage-induced negative regulation of MDM4 and potentially provide a novel means to manipulate MDM4 expression without introducing DNA damage.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. MDM4 and miR-34a are differently expressed in human cell lines.
(A) Real-time quantitative PCR was performed in quadruplicate for miR-34a using RNA extracted from the indicated cell lines before and after treatment with 0.5 ug/ml doxorubicin for 24 hours. Y-axis is log base 10. Error bars show 95% confidence intervals. Double asterisks indicate paired, one-tailed t-test values <0.01 comparing the untreated to doxorubicin treated condition for each cell line. (B) RT-qPCR as before, for MDM4.
Figure 2
Figure 2. Endogenous MDM4 is repressed by miR-34a.
(A) MCF7 cells were transfected with expression plasmids for miR-34a or a control mutant of miR-34a. Total RNA was extracted after 48 hours, and RT-qPCR was used to quantify the expression of MDM4 and the known miR-34a target genes CDK6 and CCND1. Expression is relative to the control (miR-34a-mut) transfection condition. Data are the averages of at least three independent experiments. Error bars show 95% confidence intervals. Asterisks and double asterisks indicate t-test values <0.05 and <0.01, respectively, comparing the miR-34a expression plasmid to the control mutant. (B) MCF7 cells were transfected as in (A), and whole cell lysates used for immunoblots to detect MDM4, p53, and beta-actin. Quantification relative to the 34a-mutant condition and normalized to actin are shown below each panel. (C and D) MCF7 cells were transfected with an inhibitor of miR-34a (anti-miR-34a) for 48 hours. RT-qPCR was performed for miR-34a, MDM4, and known miR-34a target genes. (E) Following transfection with miR-34a inhibitor as before, total protein was extracted and immunoblots were performed for the indicated proteins in MCF7.
Figure 3
Figure 3. The 3′ UTR of MDM4 is unresponsive to miR-34a expression.
Data are the averages of at least four independent experiments, with standard deviation indicated in error bars. Double asterisks indicate paired, one-tailed t-test values <0.01 between control and experimental conditions. Triple asterisks indicate p-value <0.001. (A) MCF7 cells were co-transfected with an expression plasmid for miR-34a or the nonfunctional mutant miR-34a-mut which produces no mature miR-34a transcript . Expression of miR-34a was determined by RT-qPCR. (B) Expression plasmids for miR-34a or miR-34a-mut were co-transfected with a luciferase reporter plasmid containing approximately 1700 bp of the MDM4 3′UTR downstream of luciferase. Cell lysates were used for luciferase assays 48 hours after transfection. Expression is relative to the expression of luciferase in the psicheck2 vector lacking a 3′ UTR. The plasmid psicheck2-AS34a is a positive control for regulation by miR-34a and contains a miR-34a response element . (C and D) As above, for H1299 cells.
Figure 4
Figure 4. The MDM4 open reading frame contains a functional miR-34a responsive site.
(A) Binding site for miR-34a predicted in human MDM4 mRNA by miRanda. Binding is indicated by solid lines, while wobble base pairing is indicated by a dashed line. Identity with the human MDM4 sequence is indicated by asterisks. The seed region of miR-34a, positions 2–7, are boxed. The homologous region of human MDM2 mRNA is also shown for comparison. (B) In MCF7 cells, the miR-34a inhibitor was cotransfected with a reporter gene containing the ORF miR-34a site from MDM4 (“Exon 11″) or the reporter with the rs79824231 SNP (“Exon 11 A>C”). Data are the averages of at least six independent experiments, with standard deviation indicated in error bars. Asterisks indicate t-test values <0.5. (C) In H1299 cells, the reporters were cotransfected with an expression plasmid for miR-34a or miR-34a-mut. Data are the averages of at least six independent experiments, with standard deviation indicated in error bars. Double asterisks indicates paired, one-tailed t-test value <0.01.
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Grants and funding

Expression plasmids for miR-34a and mutant miR-34a were kindly provided by Dr. Moshe Oren. This work was supported in part by a grant from the American Cancer Society, Ohio Division. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.