Project description:In the present study we analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs) incubated in microgravity condition, simulated by a ground-based Rotating Wall Vessel (RWV) bioreactor. Our results show that 42 miRNAs were differentially expressed in MMG-incubated PBLs compared with 1g-incubated ones. Among these, miR-9-5p, miR-9-3p, miR-155-5p, miR-150-3p, and miR-378-3p were the most dysregulated. To improve the detection of functional miRNA-mRNA pairs we performed gene expression profiles on the same samples assayed for miRNA profiling and we integrated miRNA and mRNA expression data. The functional classification of miRNA-correlated genes evidenced significant enrichments in the biological processes of immune/inflammatory response, signal transduction, regulation of response to stress, regulation of programmed cell death and regulation of cell proliferation. We identified the correlation between miR-9-3p, miR-155-5p, miR-150-3p and miR-378-3p expression with that of genes involved in immune/inflammatory response (eg. IFNG and IL17F), apoptosis (eg. PDCD4 and PTEN) and cell proliferation (eg. NKX3-1 and GADD45A). Experimental assays of cell viability and apoptosis induction validated the results obtained by bioinformatics analyses demonstrating that in human PBLs the exposure to reduced gravitational force increases the frequency of apoptosis and decreases cell proliferation. microRNA expression profiling were carried out on total RNA extracted from PBLs of twelve healthy donors at the end of 24h-incubation time in MMG and in 1g conditions. Analyses were performed by using the M-bM-^@M-^\Human miRNA Microarray kit (V2)M-bM-^@M-^] (Agilent Technologies), that allows the detection of 723 known human (miRBase v.10.1) and 76 human viral miRNAs.By comparing the expression profile of MMG-incubated vs. 1g-incubated PBLs of the same donor, we found 42 differentially expressed miRNAs, 25 up-regulated and 17 down-regulated.

Project description:Mechanisms underlying in utero fetal lung injury remain poorly defined, and a greater understanding of pathways regulating these processes may lead to novel therapies that prevent lung injury before birth. MicroRNAs (miRNAs) are small non-coding, endogenous RNAs that regulate gene expression and have been implicated in the pathogenesis of lung disease. We sought to determine whether differentially expressed miRNAs in the fetal lung following choriodecidual infection are associated with elevation of amniotic fluid (AF) cytokine levels and acute lung injury in a nonhuman primate model. After inoculating ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118-125 days gestation (term=172 days) with either Group B Streptococcus (GBS) 1 x 106 colony forming units (n=5) or saline (n=5) in the choriodecidual space, we extracted fetal lung mRNA and miRNA and profiled changes in expression. We identified 9 differentially expressed miRNAs (p<0.05, >1.5 fold change) in the GBS-exposed fetal lungs by microarray, but of these only miR-155-5p was significantly elevated by qRT-PCR (p=0.016). The microarray log2 intensity of miR-155-5p positively correlated with fetal lung injury scores and AF IL-1? and TNF-??levels (R2=0.54, p=0.016). In situ hybridization revealed that miR-155-5p is expressed throughout the fetal lung, which led us to investigate mechanisms that regulate miR-155-5p expression. Significantly elevated miR-155-5p expression was observed when immortalized human fetal airway epithelial (FeAE) cells were exposed to IL-6 and TNF-??. Overexpression of miR-155-5p in FeAE cells increased production of IL-6, CCL5/RANTES and CXCL10/IP-10. Using a luciferase reporter assay, we validated FGF9 as an authentic target of miR-155-5p, which is essential to development of the lung mesenchyme and distal epithelial branching. Collectively, these results suggest that a choriodecidual inflammatory response leads to increased AF cytokine levels, which induce miR-155-5p expression. In turn, miR-155-5p activates and recruits leukocytes (IL-6, CCL5/RANTES) and inhibits fetal lung angiogenesis (CXCL10/IP-10), meschenchymal development and epithelial branching (targets FGF9 mRNA). A therapy to antagonize miR-155-5p may ameliorate perinatal lung injury. Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118-125 days gestation (term=172 days) received choriodecidual inoculation of either: 1) Group B Streptococcus (n=5) or 2) saline (n=5). Cesarean section and fetal necropsy was performed in the first week after GBS or saline inoculation regardless of labor. RNA was extracted from fetal lungs and profiled by microarray. Results were analyzed using single gene, Gene Set, and Ingenuity Pathway Analysis. Validation was by RT-PCR and immunohistochemistry.

Project description:Mechanisms underlying in utero fetal lung injury remain poorly defined, and a greater understanding of pathways regulating these processes may lead to novel therapies that prevent lung injury before birth. MicroRNAs (miRNAs) are small non-coding, endogenous RNAs that regulate gene expression and have been implicated in the pathogenesis of lung disease. We sought to determine whether differentially expressed miRNAs in the fetal lung following choriodecidual infection are associated with elevation of amniotic fluid (AF) cytokine levels and acute lung injury in a nonhuman primate model. After inoculating ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118-125 days gestation (term=172 days) with either Group B Streptococcus (GBS) 1 x 106 colony forming units (n=5) or saline (n=5) in the choriodecidual space, we extracted fetal lung mRNA and miRNA and profiled changes in expression. We identified 9 differentially expressed miRNAs (p<0.05, >1.5 fold change) in the GBS-exposed fetal lungs by microarray, but of these only miR-155-5p was significantly elevated by qRT-PCR (p=0.016). The microarray log2 intensity of miR-155-5p positively correlated with fetal lung injury scores and AF IL-1? and TNF-??levels (R2=0.54, p=0.016). In situ hybridization revealed that miR-155-5p is expressed throughout the fetal lung, which led us to investigate mechanisms that regulate miR-155-5p expression. Significantly elevated miR-155-5p expression was observed when immortalized human fetal airway epithelial (FeAE) cells were exposed to IL-6 and TNF-??. Overexpression of miR-155-5p in FeAE cells increased production of IL-6, CCL5/RANTES and CXCL10/IP-10. Using a luciferase reporter assay, we validated FGF9 as an authentic target of miR-155-5p, which is essential to development of the lung mesenchyme and distal epithelial branching. Collectively, these results suggest that a choriodecidual inflammatory response leads to increased AF cytokine levels, which induce miR-155-5p expression. In turn, miR-155-5p activates and recruits leukocytes (IL-6, CCL5/RANTES) and inhibits fetal lung angiogenesis (CXCL10/IP-10), meschenchymal development and epithelial branching (targets FGF9 mRNA). A therapy to antagonize miR-155-5p may ameliorate perinatal lung injury.

Project description:The molecular response to hypoxia is a critical cellular process implicated in cancer, and a target for drug development. The activity of the major player, HIF1M-NM-1,M-BM- is regulated at different levels, including the transcriptional level by the Ets factor ELK3. The molecular mechanisms of this intimate transcriptional connection remain largely unknown. Whilst investigating global ELK3-chromatin interactions, we uncovered an unexpected connection that involves the microRNA hsa-miR-155-5p, a hypoxia-inducible oncomir that targets HIF1M-NM-1. One of the ELK3 chromatin binding sites, detected by Chromatin Immuno-Precipitation Sequencing (ChIP-seq) of normal Human Umbilical Vein Endothelial Cells (HUVEC), is located at the transcription start site of the MIR155HG genes that expresses hsa-miR-155-5p. We confirmed that ELK3 binds to this promoter by ChIP and QPCR. We showed that ELK3 and hsa-miR-155-5p form a double-negative regulatory loop. ELK3 depletion induced hsa-miR-155-5p expression, and hsa-miR-155-5p expression decreased ELK3 expression at the RNA level through a conserved target sequence in its 3M-bM-^@M-^Y-UTR. We further showed that the activities of hsa-miR-155-5p and ELK3 are functionally linked. Pathway analysis indicates that both factors are implicated in related processes, including cancer and angiogenesis. hsa-miR-155-5p expression and ELK3 depletion have similar effects on expression of known ELK3 target genes, and in-vitro angiogenesis and wound closure. Bioinformatic analysis of cancer RNA-seq data shows that hsa-miR-155-5p and ELK3 expression are significantly anti-correlated, as would be expected from hsa-miR-155-5p targeting ELK3 RNA. Hypoxia (0% oxygen) down-regulates ELK3 mRNA in a microRNA and hsa-miR-155-5p dependent manner. These results tie ELK3 into the hypoxia response pathway through an oncogenic microRNA and into a circuit implicated in the dynamics of the hypoxic response.M-BM- This crosstalk could be important in the development of new treatments for a range of pathologies. Examination of ELK3 DNA interactions in HUVEC cells under normal oxygen conditions

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Time-resolved RNA atlas of early human T cell activation (mRNA)

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