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Transient up-regulation of miR-155-3p by lipopolysaccharide in primary human monocyte-derived macrophages results in RISC incorporation but does not alter TNF expression

Simmonds, Rachel E. (2019) Transient up-regulation of miR-155-3p by lipopolysaccharide in primary human monocyte-derived macrophages results in RISC incorporation but does not alter TNF expression Wellcome Open Research, 4, 43.

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Background: The innate immune response is a tightly regulated process that reacts rapidly in response to pathogen-associated molecular patterns(PAMPs) such as lipopolysaccharide (LPS). Evidence is accumulating thatmicroRNAs contribute to this, although few studies have examined the earlyevents that constitute the “primary” response. LPS-dependent changes to miRNA expression were studied inMethods:primary human monocyte-derived macrophages (1°MDMs). An unbiasedscreen by microarray was validated by qPCR and a method for the absolutequantitation of miRNAs was also developed, utilising 5’ phosphorylatedRNA oligonucleotide templates. RNA immunoprecipitation was performedto explore incorporation of miRNAs into the RNA-induced silencingcomplex (RISC). The effect of miRNA functional inhibition on TNFexpression (mRNA and secretion) was investigated. Of the 197 miRNAs expressed in 1°MDMs, only five were inducedResults:>1.5-fold. The most strongly induced was miR-155-3p, the partner strand tomiR-155-5p, which are both derived from the MIR155HG/BIC gene(pri-miR-155). The abundance of miR-155-3p was induced transiently~250-fold at 2-4hrs and then returned towards baseline, mirroringpri-miR-155. Other PAMPs, IL-1β, and TNF caused similar responses.IL-10, NF-κB, and JNK inhibition reduced these responses,unlike cytokine-suppressing mycolactone. Absolute quantitation revealedthat miRNA abundance varies widely from donor-to-donor, and showed thatmiR-155-3p abundance is substantially less than miR-155-5p inunstimulated cells. However, at its peak there were 446-1,113 copies/cell,and miR-155-3p was incorporated into the RISC with an efficiency similar tomiR-16-5p and miR-155-5p. Inhibition of neither miRNA affected TNFsecretion after 2hrs in 1°MDMs, but technical challenges here are noted. Dynamic regulation of miRNAs during the primary responseConclusions:is rare, with the exception of miR-155-3p. Further work is required toestablish whether its low abundance, even at the transient peak, issufficient for biological activity and to determine whether there are specificmechanisms determining its biogenesis from miR-155 precursor.

Item Type: Article
Divisions : Faculty of Health and Medical Sciences > School of Biosciences and Medicine
Authors :
Simmonds, Rachel
Date : 6 March 2019
Funders : Wellcome Trust, Kennedy Institute Trustees
DOI : 10.12688/wellcomeopenres.15065.2
Grant Title : Wellcome Trust
Copyright Disclaimer : Copyright: © 2019 Simmonds RE. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Uncontrolled Keywords : miRNAs, miR-155, Toll-like receptor signalling, primary human cells,monocyte-derived macrophages, TNF, mycolactone
Additional Information : Embargo OK Metadata OK No Further Action
Depositing User : James Marshall
Date Deposited : 24 Aug 2020 14:00
Last Modified : 24 Aug 2020 14:00

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