Abstract
In the present study, we used high-throughput sequencing, miRNA-seq, to discover and explore the expression profiles of known and novel miRNAs in TLR ligand-stimulated vis-à-vis non-stimulated (i.e. Control) peripheral blood mononuclear cells (PBMCs) isolated from blood of healthy Murrah buffaloes. Six small RNA (sRNA) libraries were multiplexed in Ion Torrent PI chip and sequenced on Ion Proton System. The reads obtained were aligned to the Bos taurus genome (UMD3.1 assembly), which is phylogenetically closest species to buffalo (Bubalus bubalis). A total of 160 bovine miRNAs were biocomputationally identified in buffalo PBMCs and 130 putatively novel miRNAs (not enlisted in the bovine mirBase) were identified. All of these 290 miRNAs identified across the six treatment and control samples represent the repertoire of novel miRNAs for the buffalo species. The expression profiles of these miRNAs across the samples have been represented by sample dendrogram and heatmap plots. The uniquely expressed miRNAs in each treatment and control groups were identified. A few miRNAs were expressed at very high levels while the majority of them were moderately expressed. The miRNAs bta-miR-103 and -191 were found to be highly abundant and expressed in all the samples. Other abundantly expressed miRNAs include bta-miR-19b, -29b, -15a, -19a, -30d, -30b-5p and members of let family (let 7a-5p, let 7g & let 7f) in LPS and CpG treated PBMCS and bta-miR-191, -103 & -19b in Poly I:C stimulated PBMCs. Only one novel miRNA (bta-miR-11039) out of 130 identified putatively novel miRNAs, was expressed in all the six samples and differentially expressed (>2- fold) miRNAs were identified. Six of the differentially expressed miRNAs across the groups (bta-miR-421, bta-let-7i, bta-miR-138, bta-miR-21-5p, bta-miR-222 and bta-miR-27b) were subsequently confirmed by TaqMan quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, the target genes of differentially expressed miRNAs were enriched for the roles in innate immunity and TLR signaling pathways. This maiden study on profiling and cataloguing of bubaline miRNAs expressed in TLR-ligand stimulated PBMCs will provide an important reference point for future studies on regulatory roles of miRNAs in immune system of buffaloes.
Highlights
MicroRNAs are small (*22 bases), non-coding, endogenous, single-stranded RNAs that are expressed in plants, animals as well as in algae and certain viruses [1,2]
The primary aim of this study was to identify and catalogue the bubaline miRNAs from peripheral blood mononuclear cells (PBMCs) challenged with toll-like receptors (TLRs) ligands
To the extent of our knowledge, this is the first work identifying the buffalo miRNA repertoire in PBMCs challenged with the TLRligands
Summary
MicroRNAs (miRNAs) are small (*22 bases), non-coding, endogenous, single-stranded RNAs that are expressed in plants, animals as well as in algae and certain viruses [1,2]. MicroRNAs have been implicated as key regulators of immune system, development and physiological functions [3]. Increasing evidence suggests that miRNA expression plays an important role in host–pathogen interactions through modulation of both innate and acquired immune responses [4,5]. The innate immune system is activated following recognition of pathogenassociated molecular patterns (PAMPs) by host pattern recognition receptors, such as toll-like receptors (TLRs) which recognize both extracellular and intracellular PAMPs [6]. The TLRsignaling pathways are regulated by the expression of variety of miRNAs. For example, Lipopolysaccharide (LPS) treatment results in decrease of let-7 expression along with increased TLR4 expression [7]. MiR-105 and miR-19 are predicted to target mRNA encoding TLR2 leading to its decreased expression and further decrease in cytokine production in primary human keratinocytes [8]
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