Damage Associated Molecular Pattern Molecule-Induced microRNAs (DAMPmiRs) in Human Peripheral Blood Mononuclear Cells

Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA (miR) expression signature of this response is different from that of a pathogen associated molecular pattern (PAMP)-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human peripheral blood mononuclear cells (PBMCs) exposed to DAMP-containing freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6×10−4 and p<3.7×10−3), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from wildtype High Mobility Group B1 (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 expressed in PBMCs stimulated with Lipopolysaccahride (LPS) or most other Toll-like receptor (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 IKKγ mRNA, a putative target of miR-34c, increased, while protein levels of IKKγ in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1β and TNFα) decreased when PBMC cultures were briefly pre-incubated with the K+ channel (inflammasome) inhibitor, glybenclamide, suggesting that inflammasome activation is upstream of miR-34c expression in response to DAMPs. Our findings demonstrate 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.

内源性损伤相关分子模式分子（Endogenous damage associated molecular pattern molecules, DAMPs）是从坏死、受损或应激细胞中释放的物质，其与炎症反应密切相关。目前尚不明确该炎症反应的微小RNA（microRNA, miR）表达谱是否与病原体相关分子模式（pathogen associated molecular pattern, PAMP）刺激引发的炎症反应存在差异。本研究发现，在暴露于含DAMPs的冻融裂解物，或暴露于血清剥夺及葡萄糖剥夺细胞条件培养基的新鲜人外周血单个核细胞（human peripheral blood mononuclear cells, PBMCs）中，miR-34c与miR-214分别呈现显著高表达（分别为p<6×10⁻⁴与p<3.7×10⁻³）。有趣的是，与暴露于高迁移率族蛋白B1敲除（HMGB1⁻/⁻）小鼠胚胎成纤维细胞（mouse embryonic fibroblast, MEF）的裂解物或条件培养基的培养体系相比，仅miR-34c在暴露于野生型高迁移率族蛋白B1（High Mobility Group B1, HMGB1+/+）小鼠胚胎成纤维细胞的裂解物或条件培养基的PBMCs中呈现差异表达。在上述培养体系中，miR-155的表达水平极低，但在脂多糖（Lipopolysaccharide, LPS）或多数其他Toll样受体（Toll-like receptor, TLR）配体刺激的PBMCs中则显著表达，因此miR-155可作为典型的“PAMP相关miR”。暴露于受损人结直肠癌细胞系（colorectal carcinoma cell line, HCT116）的裂解物，同样可导致miR-34c与miR-214水平升高。当PBMCs预先转染抗miR-34c寡核苷酸后再暴露于裂解物时，miR-34c的推定靶标IKKγ信使RNA（mRNA）的表达水平升高；而预先转染pre-miR-34c的培养体系中，IKKγ的蛋白水平则被完全抑制。当PBMC培养体系预先用钾离子通道（炎症小体）抑制剂格列本脲（glybenclamide）进行短时间孵育时，miR-34c的表达水平（以及促炎细胞因子白细胞介素1β（IL-1β）与肿瘤坏死因子α（TNFα）的水平）均出现下降，这提示在针对DAMPs的应答中，炎症小体的激活位于miR-34c表达的上游。本研究结果表明，特定的微小RNA表达谱与受损/损伤细胞引发的炎症反应相关，这一发现对多种急性与慢性炎症性疾病具有重要研究意义。