Abstract 16544: Small Rna-sequencing Analysis of Circulating Proinflammatory Microrna Following Murine Myocardial Ischemia/reperfusion Injury

Introduction: Transient myocardial ischemic/reperfusion injury (I/R) leads to cellular RNA release into extracellular (ex) space. Ex-RNAs and certain uridine-rich miRNAs activate innate immune response via TLR7-MyD88 pathway. Systemic administration of RNase reduces myocardial inflammation and infarct size after I/R, suggesting a pivotal role of ex-RNA in I/R injury. However, the nature of plasma RNAs and their biological functions in inflammation are not well understood. Hypothesis: Multiple plasma miRNAs are elevated in response to myocardial I/R injury and some are proinflammatory. Methods: Myocardial I/R injury was created in C57BL/6 mice by thoracotomy and 45 min of left anterior descending (LAD) ligation followed by 4 h of reperfusion. Sham mice underwent same procedure without LAD ligation. Plasma RNA was extracted using Trizol LS with Cel-miR-39 as spiked-in and quantified by RNA Quant-it kits. The plasma RNA was used to create cDNA libraries for small RNA sequencing (Norgen, Canada). Three abundant miRNAs that were differentially expressed between sham and I/R groups were tested for their proinflammatory effect. Results: Myocardial I/R led to a marked increase in plasma troponin (392.1±324.6 vs. 17.9±9.9ng/ml, P<0.05) and RNA levels (163±129 vs. 81±40 ng/ml) as compared with sham mice ( Fig. A ). Plasma RNA-Seq revealed miRNA was the predominate biotype constituting of 85.7% and 79.2% of plasma RNA in sham and I/R mice, respectively ( Fig. B ). We validated three miRNA expressions using qRT-PCR and found miR191-5p, miR125a-5p, and miR125b-5p were substantially increased ( Fig. C ). When incubated with macrophage cultures, miR125a-5p and miR-125b-5p, but not miR-191-5p, induced a dose-dependent response in CXCL-2 and IL-6 productions ( Fig. D ). Conclusions: Myocardial I/R injury results in an increase in multiple miRNAs in plasma. miR125a-5p and miR125b-5p, but not miR-191-5p, are pro-inflammatory in macrophages.