FIG. 6.

miR-K10a-mediated downregulation of TWEAKR inhibits production of IL-8 and MCP-1. (A) HUVECs were transfected with miR-K10a and treated with TWEAK at 24 hpt. Culture supernatants were harvested at 24 h posttreatment and analyzed for IL-8 (top) and MCP-1 (bottom) expression. Data are expressed as the concentration of IL-8 or MCP-1 in the supernatant, and the numbers above the brackets indicate the fold change in expression upon treatment with TWEAK. Supernatants were collected from three independent experiments and analyzed in duplicate in the same assay. (B and C) HUVECs were transfected with either miR-K10a or a mutant of miR-K10a (MUTb). (B) Total cell lysates were harvested at 24 hpt and analyzed by Western blotting. Data show the average changes in normalized TWEAKR expression relative to levels in neg2-transfected cells. Averages and SD were calculated from data from three independent experiments. (C) Transfected HUVECs were treated with TWEAK at 24 hpt, and total cell RNA was harvested at 24 h posttreatment for quantitative RT-PCR analysis. Transcript levels of IFN-α, IL-6, IL-8, and MCP-1 were measured, normalized to levels of β-actin transcripts, and reported as the fold change in transcript levels upon stimulation with TWEAK. Averages and SD were calculated from data from three independent experiments. no, no miRNA.