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Expression of Human DAMPmiRs

ABSTRACT: Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA expression signature of this response is different from that of a PAMP-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human PBMCs exposed to DAMPcontaining freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6x10-4 and p<3.7x10-3), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from HMGB1+/+ mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1-/- MEFs. miR-155 expression in these cultures was negligible, but was significantly higher in PBMCs stimulated with LPS or most other TLR ligands, making it the prototypic PAMPmiR. Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKgamma mRNA, a putative target of miR-34c, increased, while protein levels of IKKgamma in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1beta and TNFalpha) decreased when PBMC cultures were briefly pre-incubated with the K+ channel (inflammasome) inhibitor, glybenclamide, suggesting that miR-34c is involved in the inflammasome pathway in response to DAMPs. Our findings suggest that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders.

ORGANISM(S): Homo sapiens

PROVIDER: GSE37399 | GEO | 2013/03/22

SECONDARY ACCESSION(S): PRJNA159895

REPOSITORIES: GEO

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Project description:Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA expression signature of this response is different from that of a PAMP-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human PBMCs exposed to DAMPcontaining freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6x10-4 and p<3.7x10-3), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from HMGB1+/+ mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1-/- MEFs. miR-155 expression in these cultures was negligible, but was significantly higher in PBMCs stimulated with LPS or most other TLR ligands, making it the prototypic PAMPmiR. Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKgamma mRNA, a putative target of miR-34c, increased, while protein levels of IKKgamma in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1beta and TNFalpha) decreased when PBMC cultures were briefly pre-incubated with the K+ channel (inflammasome) inhibitor, glybenclamide, suggesting that miR-34c is involved in the inflammasome pathway in response to DAMPs. Our findings suggest that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders. Human PBMC (peripheral blood mononuclear cells) were exposed to 4 conditions for 48 hours. In the first condition, PBMCs were exposed to conditioned media from serum-starved and glucose-deprived and heat shocked HMGB1-/- MEF cells (mouse embryonic fibroblast cells). In the second condition, PBMCs were exposed to conditioned media from serum-starved and glucose-deprived and heat shocked HMGB1+/+ MEF cells (mouse embryonic fibroblast cells). In the third condition, PBMCs were exposed to LPS (Lipopolysaccharide). In the fourth condition, the PBMCs where left untreated. Four biological repeats were done for each condition for a total of 16 samples.

Project description:Introduction: Intracerebral hemorrhage (ICH) is a severe neurological disorder with no proven treatment. microRNA (miRNA) are short, noncoding nucleic acids that are critical regulators gene expression and promising novel therapeutic targets. Our prior research of mRNA-seq in swine demonstrated that ICH increased inflammatory gene expression within six hours of onset. We now present findings from an integrated analysis of miRNA-seq and mRNA-seq in peripheral blood mononuclear cells (PBMCs) from swine ICH. We tested the hypothesis that ICH would induce miRNA within six hours and that differentially expressed (DE) inflammatory genes would be targets of the DE miRNA. Methods: In 10 pigs, one PBMC sample was collected immediately prior to ICH induction and a second six hours later; miRNA-seq and mRNA-seq were performed. To determine the miRNA that primarily regulated the DE mRNA, an aggregate score was calculated for each miRNA of interest that incorporated the combined summary statistics of its DE target mRNA. Networks of molecular interactions were generated for the target mRNA and the combined miRNA/target mRNA. Results: A total of 227 miRNA were identified with alignment to the Sus scrofa (swine) genome, and 46 were DE (FDR <0.05). When analyzed with the aggregate scoring calculation, the miRNA that were most essential for regulating the DE mRNA included the following, all of which were downregulated post-ICH: miR-29a, miR-29b, miR-31, miR-34c, miR-125a, miR-132, miR-142, miR-150, miR-151a, miR-181a, and miR-186. A highly connected network of interactions was generated from these miRNA and their target mRNA. Conclusion: ICH strongly induced numerous miRNA/mRNA targets in swine PBMCs within six hours of onset. To our knowledge, this is the first integrated analysis of miRNA-seq and mRNA-seq in any study of ICH and the first report of miRNA in swine ICH. Further research is needed to determine whether these miRNA are potential therapeutic targets, including analyzing interventions such as miRNA overexpression or inhibition

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Expression of Human DAMPmiRs

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