Integrated transcriptome analysis identifies super-regulators miRNAs and SNP-mediated allelic imbalance in pro-inflammatory macrophages.
Macrophages are the most plastic cell type of the haematopoietic system, playing a crucial role in maintaining all tissues in a healthy condition and influencing disease development and progression. This is due to their ability to sense microenvironmental signals, adopting different phenotypes in a process named “polarisation” (Mills et al., 2012). Macrophage polarisation has been extensively studied in both in vitro and in vivo models: it is not a fixed event and can be triggered by a multitude of biological and mechanical events, such as infection, tissue injury and carcinogenesis (Murray et al., 2017). In this study we investigated the expression of miRNAs and their target genes in human polarised macrophages by using an integrative approach and multiple RNA-seq experiments. We identified 9 differentially expressed miRNAs (miR-7-5p, miR-125a-3p, miR-3614-5p, miR-4773, miR-186-5p, miR-4709-3p, miR-1343-3p, miR-766-3p, miR-335-3p) which target more than 500 different genes and named them “super-regulators”. We evaluated the impact of hsa-miR-186-5p, hsa-125a-3p and hsa-155-5p by transient transfection of miRNA mimics on macrophage transcriptomes and observed genetic signatures that recapitulate the M1-like phenotype. Our results demonstrate the existence of a macrophage-specific targetome that underlie most biological pathways involved in inflammation. Experiment design PBMCs were isolated from 80mL of peripheral venous blood using Ficoll centrifugation. CD14+ monocytes were positively selected using anti-CD14 beads. Monocytes were cultured for 7 days (typically in a 6 well plate, 1 million cells per well) in RPMI-1640 (Gibco) plus 10% (v/v) low endotoxin heat inactivated FBS (PanBiotech), 1% (v/v) L–glutamine (Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). Recombinant human (rh) M–CSF (100 ng/ml, Peprotech/Immunotools) was added to the media to allow the differentiation of monocytes into macrophages. After 7 days of differentiation, monocyte-derived macrophages were transiently transfected with 50nM of miR-155-5p, miR-125a-3p or miR-186-5p mimics and a negative control (Horizon Discovery) for 24 hours. Transient transfection was performed using Viromer Green (Lipocalyx). Total RNA was extracted by using the miRNeasy Mini Kit (Qiagen) according to manufacturer instructions.
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RNA sequencing experiments were performed by the genome sequencing company Novogene Co. Ldt (https://en.novogene.com), using Illumina platform (NovaSeq platform, paired-end 150 bp). The quality of samples were checked using FastQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (2015)). Low quality reads and adapters were trimmed using trimmomatic algorithm (Anthony M. Bolger,2014) with the default parameter. Samples were further processed and quantified with Salmon (Patro,2017) using the human reference transcriptome (Ensemble 93).