Fig. 3

miR-574-5p overexpression stimulates whereas miR-574-5p knockdown suppresses interferon and cytokine responses in human cells. NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, compared to an appropriate group. (a) Verification of miR-574-5p overexpression or knockdown in THP1 cells (n = 3–4). (b) miR-574-5p overexpression stimulated whereas miR-574-5p knockdown suppressed NFκB and interferon-mediated transcriptional activities in THP1 monocytic cells. THP1 cells were co-transfected with plasmids as indicated and incubated for 24 h. Cells were harvested for the luciferase reporter assays and β-galactosidase activity assay (n = 3). (c) Effects of miR-574-5p overexpression or knockdown on the protein expression of myeloid differentiation primary response gene-88 (MyD88), TNF receptor-associated factor-3 (TRAF3), STAT1 and pSTAT1^Y701 in THP1 cells. THP1 cells were transfected with indicated plasmids for 24 h, or infected with lentiviruses as indicated for 96 h. Cells were harvested for western blotting assays (n = 3). (d-e) miR-574-5p exposure potently stimulated the secretion of IFNα/γ, TNFα and IL6 in human PBMCs (n = 3). About 1 × 10⁶ human PBMCs were seeded in 6-well plate and treated with 1 µg/ml R848 or 10 µg/ml of Dotap-PS-miR-16 or Dotap-PS-miR-574-5p for 24 h. Alternatively, human PBMCs were infected with LV-MIR-ctrl or LV-hsa-MIR574 for 96 h. Subsequently, the media were harvested for ELISA analyses. (f) Dotap-formulated miR-574-5p transfection stimulated NFκB transcriptional activity in HEK-Blue-TLR8 cells but not in HEK-Blue-TLR7 cells. HEK-Blue-TLR7 or HEK-Blue-TLR8 cells were grown and co-transfected with a luciferase reporter plasmid (pNFκB-luc) and pSV40-β-galactosidase (3:1). 24 h after the transfection, cells were stimulated with 1 µg/ml of R848 or 10 µg/ml of Dotap-PS-miR-16 or Dotap-PS-miR-574-5p and in the absence or presence of 4 mM of uridine for 8 h. Cells were collected for luciferase activity assay (n = 3)