The NK Cell MicroRNA Transcriptome Defined by Next-Generation Sequencing Identifies IL-15-Signaled Alterations In Mature MiR-223 Expression, and MiR-223 as a Potential Regulator of Murine Granzyme B
Authors: Fehniger Todd A , Wylie Todd , Germino Elizabeth , Leong Jeffrey W , Magrini Vincent J , Koul Sunita , Keppel Catherine R , Schneider Stephanie E , Koboldt Daniel C , Sullivan Ryan P , Heinz Michael E , Crosby Seth D , Nagarajan Rakesh , Ramsingh Giridharan , Link Daniel C , Ley Timothy J , Mardis Elaine R 
Published: October 2019

Abstract
Abstract 104
Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g. granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes has been established, little is known about miRNAs in NK cells. Here, we utilized two next-generation sequencing platforms, Illumina GA and SOLiD, to define the miRNA transcriptomes of resting and IL-15-activated primary murine NK cells. Using Illumina GA sequencing, we identified expression of 297 mature miRNA and 64 miRNA* sequences in NK cells at read counts of 1 to 507,369. The 5 miRNAs with the highest abundance (percent normalized read count) in resting NK cells were miR-21 (31.6%), miR-16 (8.3%), miR-142-5p (6.5%), miR-142-3p (3.8%), miR-24 (3.7%). Detection of known miRNA was similar using SOLiD sequencing in both resting (r2=0.79) and IL-15-activated (r2=0.75) NK cells, with most of the discordant detection arising in miRNAs with low expression (1-10 read counts). These results were further validated using real-time RT-qPCR and microarray profiling. A bioinformatics pipeline was developed that identified 37 novel miRNA genes that encoded 26 novel mature miRNA sequences, including miRNAs processed from snoRNAs. Of 84 miRNAs with significantly altered expression levels (Illumina, normalized read counts) following IL-15-activation, miR-223 demonstrated the greatest fold decrease (2.3, P=1.03E-10). The decrease in mature miR-223 following 24 hours of IL-15 activation was confirmed in SOLiD sequencing (7.7 fold-decrease, P=1.0E-10), qPCR (2.5 fold-decrease, P=0.01), and microarrays (4.6 fold-decrease, P=0.003). IL-15 induced reduction in mature miR-223 was time-dependent with fold decreases of 1.5 (NS) at 8 hours, 2.2 (P=0.003) at 24 hours, and 8.1 (P=0.012) at 48 hours, assessed by real-time qPCR. Decreases in mature miR-223 expression early following activation likely occur via regulation of the miRNA processing machinery, since primary and precursor miR-223 transcripts exhibited increased expression after 8 hours (2.1±0.3 fold increase, P=0.03) and 24 hours (2.5±0.3 fold increase, P=0.03) of IL-15-activation. MiR-223 is computationally predicted to bind to the murine GzmB 3'UTR sequence, and we therefore evaluated the ability of miR-223 to target the murine GzmB 3'UTR in vitro. Overexpression of miR-223 in 293T cells decreased luciferase protein signal controlled by the GzmB 3'UTR by 44±2% (P=0.002) without affecting negative controls (perforin 3'UTR or no 3'UTR). Moreover, mutating the miR-223 predicted binding site within the murine GzmB 3'UTR abrogated miR-223 mediated regulation. These data suggest that miR-223 directly targets the murine GzmB 3'UTR, and thereby contributes to tonic suppression of GzmB mRNA translation in resting NK cells. Thus, the sequenced and validated miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology.

Disclosures:
No relevant conflicts of interest to declare.