Genome-wide analysis of human neutrophils mirnome identified miR-23a as a critical regulator of the apoptotic Fas antigen expression

Over the past decade, genome-wide studies have made it clear that the mammalian genome is pervasively transcribed and this led to the identification of non-protein coding RNA molecules. Only 2% of the mammalian genome accounts for protein-coding sequences, so that the majority is transcribed as noncoding RNA (ncRNA). ncRNAs appear to be highly conserved and are considered to be an important component for genetic regulation via epigenetic mechanisms. The regulatory ncRNAs can be classified into long non-coding RNAs (lncRNAs) and microRNAs (miRNAs). miRNAs increasingly recognized to play a pivotal role in both physiological and pathological conditions, including mammalian development, cardiovascular, neurodegenerative and metabolic diseases, cancer and immune disorders. Despite the evidence for the importance of miRNAs in immune cell function and development, very limited information is available regarding how miRNAs regulate neutrophils development, lifespan and functions. Based on these premises, the purpose of this study was to provide a comprehensive analysis of microRNAs expression profile and to identify their role in primary human neutrophils under resting and stimulatory conditions. To achieve this goal we performed whole transcriptome analysis and characterized the pri-miRNome of resting and LPS-stimulated neutrophils. In parallel, the same analysis was performed also on autologous monocytes, in order to get insight into cell-specific miRNA profile. The results of this study showed that TLR4 engagement triggers the transcription of 37 pri-miRNAs. Additionally, comparison of LPS-induced pri-miRNA expression profile of neutrophils with that of autologous monocytes identified subsets of pri-miRNAs modulated by LPS in both cell type in a cell-specific manner. LPS induced the expression of twelve pri-miRNAs in neutrophils, among which we further validated the expression of the mature forms of the miR23a cluster members. The members of this cluster have been described to play important roles in various biological and pathological processes, but their role has never been previously identified in resting and/or activated neutrophils. Upon LPS stimulation neutrophils upregulate only the mature forms of miR-23a-5p and miR-27a-5p. In silico target prediction indicated Fas, a death receptor mediating cell apoptosis, among the miR-23a predicted target genes. Herein we provide several experimental proofs demonstrating that LPS reduces neutrophil Fas-induced apoptosis in a miR-23a-dependent manner. In fact, and consistent with a prolonged neutrophils’ half-life, in the presence of LPS a parallel upregulation of miR-23a and down-regulation of Fas membrane protein, but not Fas mRNA, expression was detected, suggesting involvement of a post-transcriptional regulation for Fas. Both Fas mRNA and miR-23a were detected in Ago immunoprecipitates only in LPS-stimulated neutrophils, thus indicating that LPS promotes miR-mediated post-transcriptional silencing of FAS. Finally, neutrophils overexpressing miR-23a reduced Fas membrane receptor. Taken together these data demonstrated that LPS stimulation affects neutrophil apoptosis via increasing miR-23a, which in return decreases the Fas expression on neutrophil surface. Collectively, we have described the expression profile of pri-miRNAs in resting and LPS-activated human neutrophils and their autologous monocytes, resulting in identification of cell type specific pri-miRNAs. The information from this whole transcriptome study led us to link the LPS to neutrophil apoptosis via miRNA modulation. Our data could be substantially used for further investigation on the role of miRNAs in neutrophils biology.