Abstract WP319: miRNA Screening of Natural Killer Cell Identifies miR-1224 as a Post-Transcriptional Regulator of NK Cell Function After Ischemic Stroke

Abstract

Objectives: To screen miRNA profile of peripheral NK cells in ischemic stroke mouse model and investigate a most promising candidate (miR-1224) for post-transcriptional regulation of NK cell function after ischemic stroke. Methods: Mice were subjected to a 60 min focal cerebral ischemia produced by transient intraluminal occlusion of MCAO. For NK cell isolation, cell suspensions from the spleens after reperfusion were enriched for NK cells using magnetic-bead sorting system after staining with anti-NK1.1 microbeads. The nCounter Mouse miRNA array was used to analyze miRNA expression profile in splenic NK cells over the time course of experimental ischemic stroke. Based on the miRNA data, we further in vitro modulated miR-1224 in NK cells using mimics or inhibitor, then injected i.v into Rag2-/-γc-/- recipient mice. Neurological function score was compared and spontaneous infection was assessed by pulmonary bacteria colony culture, and changes in potential signaling pathway (SP1/TNF-α) were verified by rt-PCR and western blot. Results: Through miRNA expression profile analysis, we have identified significant changes at each time point in peripheral NK cells after cerebral ischemia. Among all screened miRNA, miR-1224 remarkably increased in MCAO group, which was verified by PCR. Then isolated NK cells treated with mimics or inhibitors, were transferred to Rag2-/-γc-/- recipient mice. Compared with WT mice, Rag2-/-γc-/- mice with miR-1224 inhibitor exhibited increased NK cell number, enhanced NK cell activation/cytotoxicity feature, as well as better neurological behaviors and reduced pulmonary infection after MCAO. Moreover, compared with the control group, NK cells with miR-1224 inhibitor showed significantly increased SP1 gene and protein phosphorylation. As SP1 gene is one of the potential targets of miR-1224, this study suggests that miR-1224 may regulate NK cell function after MCAO, which is associated with SP1 pathway. Conclusion: The miRNA profiling of splenic NK cells provided insight into the functional mechanism and signaling pathways underlying the distinct organ-specific NK cell properties, which will contribute to the better understanding of NK cell mediated immune-response in relation to different stages of stroke.