Study of MicroRNA-34a mediated post transcriptional regulation of MDM4

MDM4 is an important negative regulator of the tumor suppressor p53. In normal unstressed cells the activity of p53 is kept under control by MDM4 and its homologue MDM2. MDM4 is said to possess oncogenic potential based on the evidence of its overexpression in many cancers. Until recently it was believed MDM4 is constitutively transcribed; however a decrease in full length MDM4 in response to genotoxic stress was observed paving way for exploring the mechanism responsible for this. It was observed miR-34a a member of the miR34 family which is a direct transcriptional targets of p53 could have a potential role in regulation of MDM4 expression. The 3\u27untranslated region of MDM4 was also seen to contain several miR-34a binding sites. However reporter assays with select regions of the 3\u27UTR revealed that the 3\u27UTR was unresponsive to miR-34a mediated regulation. Reassessment of the MDM4 gene revealed presence of a potential miR-34a regulatory site in the protein coding exon eleven of MDM4. This site was further considered to check for functionality in response to miR-34a modulation. A reporter with the miR-34a site from the coding region was constructed. This reporter was responsive to overexpression or inhibition of endogenous miR-34a in H1299 and MCF7 cells respectively ascertaining the functionality of this site. A SNP leading to an A to C transversion in the seed region of this miR-34a site in the exon 11 was predicted to disrupt responsiveness to miR-34a. We confirmed this by creating point mutants and performing reporter assays. This study was designed to understand the regulation of MDM4 in absence of DNA damage conditions. Understanding the role of miR-34a in regulation of MDM4 will pave way for designing specific therapeutic strategy for reactivation of p53 via inhibition of MDM4 in cancer that overexpress MDM4 and retain wild type p53

MicroRNA-34a Modulates MDM4 Expression via a Target Site in the Open Reading Frame

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

MicroRNA-34a Modulates MDM4 Expression via a Target Site in the Open Reading Frame

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

Low-Dose Oxygen Enhances Macrophage-Derived Bacterial Clearance following Cigarette Smoke Exposure

Background. Chronic obstructive pulmonary disease (COPD) is a common, smoking-related lung disease. Patients with COPD frequently suffer disease exacerbations induced by bacterial respiratory infections, suggestive of impaired innate immunity. Low-dose oxygen is a mainstay of therapy during COPD exacerbations; yet we understand little about whether oxygen can modulate the effects of cigarette smoke on lung immunity. Methods. Wild-type mice were exposed to cigarette smoke for 5 weeks, followed by intratracheal instillation of Pseudomonas aeruginosa (PAO1) and 21% or 35–40% oxygen. After two days, lungs were harvested for PAO1 CFUs, and bronchoalveolar fluid was sampled for inflammatory markers. In culture, macrophages were exposed to cigarette smoke and oxygen (40%) for 24 hours and then incubated with PAO1, followed by quantification of bacterial phagocytosis and inflammatory markers. Results. Mice exposed to 35–40% oxygen after cigarette smoke and PAO1 had improved survival and reduced lung CFUs and inflammation. Macrophages from these mice expressed less TNF-α and more scavenger receptors. In culture, macrophages exposed to cigarette smoke and oxygen also demonstrated decreased TNF-α secretion and enhanced phagocytosis of PAO1 bacteria. Conclusions. Our findings demonstrate a novel, protective role for low-dose oxygen following cigarette smoke and bacteria exposure that may be mediated by enhanced macrophage phagocytosis

The MDM4 open reading frame contains a functional miR-34a responsive site.

<p>(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.</p

The 3′ UTR of MDM4 is unresponsive to miR-34a expression.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042034#pone.0042034-RaverShapira1" target="_blank">[21]</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042034#pone.0042034-Lal1" target="_blank">[51]</a>. (C and D) As above, for H1299 cells.</p

MDM4 and miR-34a are differently expressed in human cell lines.

<p>(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.</p

Endogenous MDM4 is repressed by miR-34a.

<p>(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.</p

HIV Impairs Lung Epithelial Integrity and Enters the Epithelium to Promote Chronic Lung Inflammation.

Several clinical studies show that individuals with HIV are at an increased risk for worsened lung function and for the development of COPD, although the mechanism underlying this increased susceptibility is poorly understood. The airway epithelium, situated at the interface between the external environment and the lung parenchyma, acts as a physical and immunological barrier that secretes mucins and cytokines in response to noxious stimuli which can contribute to the pathobiology of chronic obstructive pulmonary disease (COPD). We sought to determine the effects of HIV on the lung epithelium. We grew primary normal human bronchial epithelial (NHBE) cells and primary lung epithelial cells isolated from bronchial brushings of patients to confluence and allowed them to differentiate at an air- liquid interface (ALI) to assess the effects of HIV on the lung epithelium. We assessed changes in monolayer permeability as well as the expression of E-cadherin and inflammatory modulators to determine the effect of HIV on the lung epithelium. We measured E-cadherin protein abundance in patients with HIV compared to normal controls. Cell associated HIV RNA and DNA were quantified and the p24 viral antigen was measured in culture supernatant. Surprisingly, X4, not R5, tropic virus decreased expression of E-cadherin and increased monolayer permeability. While there was some transcriptional regulation of E-cadherin, there was significant increase in lysosome-mediated protein degradation in cells exposed to X4 tropic HIV. Interaction with CXCR4 and viral fusion with the epithelial cell were required to induce the epithelial changes. X4 tropic virus was able to enter the airway epithelial cells but not replicate in these cells, while R5 tropic viruses did not enter the epithelial cells. Significantly, X4 tropic HIV induced the expression of intercellular adhesion molecule-1 (ICAM-1) and activated extracellular signal-regulated kinase (ERK). We demonstrate that HIV can enter airway epithelial cells and alter their function by impairing cell-cell adhesion and increasing the expression of inflammatory mediators. These observed changes may contribute local inflammation, which can lead to lung function decline and increased susceptibility to COPD in HIV patients