MicroRNAs Implicated in Dysregulation of Gene Expression Following Human Lung Transplantation

Background: Lung transplantation remains the only viable treatment option for the majority of patients with advanced lung diseases. However, 5-year post-transplant survival rates remain low primarily secondary to chronic rejection. Novel insights from global gene expression profiles may provide molecular phenotypes and therapeutic targets to improve outcomes after lung transplantation. Methods: Whole-genome gene expression profiling was performed in a cohort of patients that underwent lung transplantation as well as healthy controls using the Affymetrix Human Exon 1.0ST Array. To explore the potential roles of microRNAs (miRNAs) in regulating lung transplantation-associated gene dysregulation, miRNA expression levels were also profiled in the same samples using the Exiqon miRCURY™ LNA Array. Results: In a cohort of 18 lung transplant patients, 364 dysregulated genes were identified in Caucasian lung transplant patients relative to normal individuals. Pathway enrichment analysis of the dysregulated genes pointed to Gene Ontology biological processes such as "defense response", "immune response” and "response to wounding”. We then compared the expression profiles of potential regulating miRNAs, suggesting that dysregulation of a number of lung transplantation-associated genes (e.g., ATR, FUT8, LRRC8B, NFKBIA) may be attributed to the dysregulation of their respective regulating miRNAs. Conclusions: Following human lung transplantation, a substantial proportion of genes, particularly those genes involved in certain biological processes like immune response, were dysregulated in patients relative to their healthy counterparts. This exploratory analysis of the relationships between miRNAs and their gene targets in the context of 3 lung transplantation warrants further investigation and may serve as novel therapeutic targets in lung transplant complications

<i>GADD45a</i> Promoter Regulation by a Functional Genetic Variant Associated with Acute Lung Injury

<div><p>Rationale</p><p>Growth arrest DNA damage inducible alpha (<i>GADD45a</i>) is a stress-induced gene we have shown to participate in the pathophysiology of ventilator-induced lung injury (VILI) via regulation of mechanical stress-induced Akt ubiquitination and phosphorylation. The regulation of <i>GADD45a</i> expression by mechanical stress and its relationship with acute lung injury (ALI) susceptibility and severity, however, remains unknown.</p><p>Objectives</p><p>We examined mechanical stress-dependent regulatory elements (MSRE) in the <i>GADD45a</i> promoter and the contribution of promoter polymorphisms in <i>GADD45a</i> expression and ALI susceptibility.</p><p>Methods and Results</p><p>Initial studies in <i>GADD45a</i> knockout and heterozygous mice confirmed the relationship of <i>GADD45a</i> gene dose to VILI severity. Human lung endothelial cells (EC) transfected with a luciferase vector containing the full length <i>GADD45a</i> promoter sequence (−771 to +223) demonstrated a >4 fold increase in <i>GADD45a</i> expression in response to 18% cyclic stretch (CS, 4 h) compared to static controls while specific promoter regions harboring CS-dependent MSRE were identified using vectors containing serial deletion constructs of the <i>GADD45a</i> promoter. <i>In silico</i> analyses of <i>GADD45a</i> promoter region (−371 to −133) revealed a potential binding site for specificity protein 1 (SP1), a finding supported by confirmed SP1 binding with the <i>GADD45a</i> promoter and by the significant attenuation of CS-dependent <i>GADD45a</i> promoter activity in response to SP1 silencing. Separately, case-control association studies revealed a significant association of a <i>GADD45a</i> promoter SNP at −589 (rs581000, G>C) with reduced ALI susceptibility. Subsequently, we found allelic variation of this SNP is associated with both differential GADD45a expression in mechanically stressed EC (18% CS, 4 h) and differential binding site of interferon regulatory factor 7 (IRF7) at this site.</p><p>Conclusion</p><p>These results strongly support a functional role for <i>GADD45a</i> in ALI/VILI and identify a specific gene variant that confers risk for ALI.</p></div

<i>GADD45a</i> promoter SNP rs581000 genotypes in Chicago and Spanish cohorts.

<p>AA represents African American and EA represents European American in the Chicago study.</p

<i>GADD45a</i> promoter activity and functional promoter region in response to mechanical stress.

<p>(<b>A</b>) Human pulmonary artery endothelial cells (EC) were plated on Bioflex stretch plates and transfected with a full-length <i>GADD45a</i> promoter vector followed by cyclic stretch (CS, 5% or 18%) for 4h. Dual luciferase reporter assay revealed about significant increases in <i>GADD45a</i> promoter activity proportional to the degree of CS (n = 3/condition, * p<0.05 compared to respective static controls). (<b>B</b>) Schematic representation of full-length and deletion fragments of <i>GADD45a</i> promoter and empty pSGG reporter vector. Promoter fragments with 200 bp serial deletions were PCR amplified and cloned into empty pSGG vector to generate <i>GADD45a</i> promoter constructs. (<b>C</b>) EC were transfected with <i>GADD45a</i> promoter deletion constructs followed by 18% CS (4 h). Dual luciferase reporter assay revealed ∼4 fold increase in reporter activity in both full-length (1.01 kb) and 0.6 kb (−371 to +237) <i>GADD45a</i> promoter fragment in response to 18% CS cells compared to respective static controls. Comparatively, 18% CS-induced luciferase activity was significantly decreased in 0.8 kb, 0.4 kb and 0.2 kb fragments. (<b>D</b>) <i>In silico</i> analysis of <i>GADD45a</i> promoter region −371 to −133 by Genomatix predicted binding sites for transcription factor SP1. The sequence logo graphically represents the SP1 consensus sequence. The relative height of each base within each stack represents relative frequency of the corresponding base at that position. Highly conserved positions are represented by higher stacks of base symbols A, T, G, C. (<b>E</b>) The binding of SP1 with the <i>GADD45a</i> promoter was detected by EMSA using biotin-labeled <i>GADD45a</i> promoter fragment and HL60 nuclear extract in the presence or absence of a non-labeled SP1 competitor and antibodies specific for SP1. DNA-protein interaction was characterized by complex formation upon the addition of nuclear proteins, which was blocked in the presence of an SP1 competitor. Addition of SP1 antibody altered the mobility of the complex, characterized by a super-shift of DNA-protein complex (arrow). (<b>F</b>) EC co-transfected with SP1 siRNA and a full-length <i>GADD45a</i> promoter vector followed by 18% CS (4 h) exhibited significantly attenuated <i>GADD45a</i> promoter activity compared to unsilenced cells transfected with the full-length <i>GADD45a</i> promoter vector alone (n = 3/condition, * p<0.05). Silencing was confirmed by Western blotting (representative blots shown).</p

<i>GADD45a</i> SNP association with severe sepsis and ALI.

<p>(<b>A</b>) Plots of association <i>P</i> values of tested <i>GADD45a</i> SNPs identified significant association of promoter SNP rs581000 with ALI in AA of Chicago cohort and with both severe sepsis and ALI in Spanish cohort. In Spanish cohort intergenic SNP rs607375 was associated with both severe sepsis and ALI. The dashed line represents a <i>p</i>-value of 0.05 and a schematic of the <i>Gadd45a</i> gene structure below indicates relative position of SNPs tested for association. Black boxes within the gene schematic represent coding exons. White boxes represent the 5′ and 3′ UTR, respectively. (<b>B</b>) Panel represents linkage disequilibrium (LD) patterns across the genotyped SNPs in the two cohorts. High LD is noted between promoter SNP rs581000 and rs607375 in both EAs from the Chicago cohort and in the Spanish cohort. Each diamond of the LD plot represents a pairwise SNP comparison. Numbers and colors in each diamond indicate the magnitude of LD between pairs of SNPs (D′ = 100 corresponds to complete LD denoted by red; D′ = 0 corresponds to absence of LD denoted by white; blue represents an intermediate association).</p

GADD45a expression and mechanical stress-induced murine lung injury.

<p>(<b>A</b>) <i>GADD45a</i> mRNA levels from lung homogenates of wildtype (WT) mice subjected to high tidal volume mechanical ventilation (V_T = 40 ml/kg, 4 h) were significantly increased in comparison to spontaneously breathing (SB) control mice (n = 3/group, *p<0.05). (<b>B</b>) WT, <i>GADD45a</i> heterozygous (<i>GADD45a</i>^+/−), and <i>GADD45a</i> knockout (<i>GADD45a^−/−</i>) mice were subjected to high tidal volume mechanical ventilation (V_T 40 ml/kg, 4 h) and BAL fluid was collected for analyses. BAL fluid total protein levels were significantly higher in <i>GADD45a</i>^+/− mice compared to WT and significantly less than <i>GADD45a^−/−</i> mice. SB animals showed no difference (n = 3/group, *p<0.05). (<b>C</b>) Total cell counts in BAL fluid from VILI-challenged <i>GADD45a^−/−</i> mice were significantly increased compared to both <i>GADD45a^+/−</i> and WT animals while there was no difference was observed between WT and <i>GADD45a</i>^+/− animals after VILI challenge (n = 3/group, *p<0.05).</p

Summary of SNPs in <i>GADD45a</i> gene.

a<p>Position on chromosome 1 according to NCBI Build 37.</p>b<p>Location relative to reference sequence NM_001924.3.</p>c<p>Represents minor allele frequency in gene resequencing data in 15 African American (AA) and 15 European American (EA).</p><p>Boldface highlights SNPs genotyped in both Chicago and Spanish study.</p><p>** Represents tagging SNP.</p><p>* Represents 3′UTR region.</p><p># represents synonymous SNP (Glu164Glu).</p

Population characteristics of Chicago and Spanish samples studied.

<p>*expressed as mean ± SD.</p><p>APACHE II: Acute Physiology and Chronic Health Evaluation.</p><p># Male/Female.</p><p>- Data not available.</p

Promoter SNP rs581000 effect on mechanical stress-induced <i>GADD45a</i> activity.

<p>(<b>A</b>) EC were transfected with rs581000_C or rs581000_G vectors and then subjected to 18% CS (4 h). Cells transfected with rs581000_C vector demonstrated significantly increased luciferase reporter activity in response to CS compared to rs581000_G. (<b>B</b>) EMSA was performed with nuclear extracts from HeLa cells and biotin-labeled <i>GADD45a</i> promoter fragments with either the C allele or G allele in the presence or absence of an interferon regulatory factor 7 (IRF7) competitor. Binding affinity of the allelic variants with nuclear proteins is indicated by arrows (arrows = protein-bound DNA). (<b>C</b>) Schematic representation of <i>GADD45a</i> promoter regions with regulatory elements. SNP rs581000 creating c<i>is</i>-regulatory element IRF7 binding site, in the region −771 to −571 are associated with enhancement of promoter activity that overrides putative transcription suppressors in the region −571 to −371 and contributes to regulatory element in the region −371 to −133. Consistent with the deletion of the region −771 to −571 results in significant reduction of promoter activity but is restored by further deletion of region −571 to −371.</p

Lung Transcriptomics during Protective Ventilatory Support in Sepsis-Induced Acute Lung Injury

<div><p>Acute lung injury (ALI) is a severe inflammatory process of the lung. The only proven life-saving support is mechanical ventilation (MV) using low tidal volumes (LVT) plus moderate to high levels of positive end-expiratory pressure (PEEP). However, it is currently unknown how they exert the protective effects. To identify the molecular mechanisms modulated by protective MV, this study reports transcriptomic analyses based on microarray and microRNA sequencing in lung tissues from a clinically relevant animal model of sepsis-induced ALI. Sepsis was induced by cecal ligation and puncture (CLP) in male Sprague-Dawley rats. At 24 hours post-CLP, septic animals were randomized to three ventilatory strategies: spontaneous breathing, LVT (6 ml/kg) plus 10 cmH₂O PEEP and high tidal volume (HVT, 20 ml/kg) plus 2 cmH₂O PEEP. Healthy, non-septic, non-ventilated animals served as controls. After 4 hours of ventilation, lung samples were obtained for histological examination and gene expression analysis using microarray and microRNA sequencing. Validations were assessed using parallel analyses on existing publicly available genome-wide association study findings and transcriptomic human data. The catalogue of deregulated processes differed among experimental groups. The ‘response to microorganisms’ was the most prominent biological process in septic, non-ventilated and in HVT animals. Unexpectedly, the ‘neuron projection morphogenesis’ process was one of the most significantly deregulated in LVT. Further support for the key role of the latter process was obtained by microRNA studies, as four species targeting many of its genes (Mir-27a, Mir-103, Mir-17-5p and Mir-130a) were found deregulated. Additional analyses revealed '<i>VEGF</i> signaling' as a central underlying response mechanism to all the septic groups (spontaneously breathing or mechanically ventilated). Based on this data, we conclude that a co-deregulation of '<i>VEGF</i> signaling' along with 'neuron projection morphogenesis', which have been never anticipated in ALI pathogenesis, promotes lung-protective effects of LVT with high levels of PEEP.</p></div