The purpose of our study was to describe the expression profiles of primary and mature forms of microRNAs (pri-miRs and miRs) in freshly isolated human polymorphonuclear neutrophils (PMN) as well as cultured cells under resting and stimulatory conditions. Human PMNs were purified from buffy coats and cultured in presence or absence of LPS for 1.5 and 4 hours. RNA-seq analysis was conducted using the human transcriptome as the reference (Ensembl 77). DESeq2 was used for differential transcript expression analysis. Pri-miRs and miRs differentially expressed were validated by RT-qPCR and Individual TaqMan miRNA Assay, respectively. miRanda and TargetScan miRNA target prediction algorithms were used to identify the predicted miRs target genes. The FatiGO tool of the Babelomics was used to identify significant over-representation of functional annotation for the predicted miR target genes. Our data show that 381 pri-miRs are constitutively expressed in circulating PMNs. Culturing PMN modulates the expression of 59 pri-miRs, which the modulation is not affected by the presence of LPS. The expression of 13 pri-miRs was increased in LPS-stimulated PMNs as compared to 4h cultured cells. The increase in expression of the mature forms of 3 pri-miRs (miR-23a, miR24-2 and miR27a) was validated. Based on the evidence that the main function of a miR is destabilizing its target mRNA, the mRNA subset with decrease in their expression upon LPS-stimulation was extracted from RNAseq data. Subsequently these mRNAs were analyzed by target prediction algorithms. We found 425 mRNA transcripts to be potential targets of the 3 miRs with increase expression pattern. Moreover GO term analysis revealed that kinase activity was over-represented among targeted mRNAs. In conclusion, this study provides the first comprehensive miR expression profile of resting and LPS-activated human PMN, which serves as a reference for further research on PMN related abnormalities.
|Titolo:||MicroRNA profiling in human neutrophils during activation|
|Data di pubblicazione:||2016|
|Appare nelle tipologie:||04.02 Abstract in Atti di convegno|