Effect Of Dicer On Macrophage Polarization

Objective: To analyze Dicer expression in macrophage after stimulation withwell-established M1or M2polarizing agents; Inhibit Dicer expression in macrophage byRNA interference based on lentivirus delivery in vitro and observe the changes of ofmacrophage polarization when Dicer were down-regulated, hoping to identify Dicer aswhat we believe to be a regulator of macrophage polarization.Methods:(1) To determine whether Dicer are critical in macrophage polarization, wefirst performed quantitative RTPCR and western blot to analysis for Dicer in mouseperitoneal macrophages (PMs), M-CSF–differentiated mouse bone marrow–derivedmacrophages (BMDMs) and mouse macrophage cell line RAW264.7after stimulation withwell-established M1or M2polarizing agents.(2) We designed and constructed siRNAaimed Dicer gene, and loaded them into pFIV-based Lentivector Expression System whichhas both U6and H1promoters. Transfected them into mouse macrophage cells lineRAW264.7, detected the Dicer expression by Real time PCR and Western blot, detected themir-155, mir-223expression by Real time PCR, detected the change of hyperplasy by CCK8,the apoptosis by follow cytometry analysis, the chemotactic activity by transwell, thephagotytosis ability by FITC-dextran.(3) Performed quantitative RTPCR to observe thechanges of M1or M2marker gene expression after stimulation with M1or M2polarizingagents in Dicer-deficient macrophages; Incubated macrophages with E. coli, S. aureus and L.monocytogenes to determine the bactericidal function of Dicer-deficient macrophages.Results:(1) Dicer expression is augmented by M2and inhibited by M1stimuli whileincreased when LPS-tolerance in macrophages.(2) Construct Dicer-specific siRNALentivector expression vectors and efficiently inhibit the expression of Dicer and mir-155,mir-223in mouse macrophage cells line RAW264.7. When Dicer was down-regulated, theproliferation, phagotytosis and chemotactic activity were not changed, while the apoptosiswas increased in macrophages.(3) Dicer deficiency enhances macrophage M1polarizationwhile inhibited M2marker gene expression; Dicer-deficient macrophages exhibit enhancedbactericidal activity. Conclusion：(1) Dicer expression is regulated by macrophage polarization. Dicerexpression was robustly induced in M2macrophages and reduced in M1macrophages whileincreased when LPS-tolerance;(2) Dicer deficiency increased macrophages apoptosis, whilehad no effect to other functions;(3) Dicer-deficient macrophages demonstrated decreasedanti-inflammatory (M2) gene expression, increased proinflammatory (M1) gene expressionand enhanced bactericidal activity. Collectively, these data identify Dicer as what we believeto be a regulator of macrophage polarization.