Figure 7.

MiR-125a-5p and let-7e-5p directly target TLR4. (A) Wild-type or MRE-mutated luciferase constructs based on the 3′UTR of TLR4 were cotransfected in HEK-293T cells with mimics of miR-125a-5p, let-7e-5p, miR-99b-5p, or a negative control (CT). Results are expressed as mean (% variation ± SEM; n = 6) of the ratio between renilla luciferase and firefly control luciferase activities. (B,C) Cell extracts from THP-1 cells transduced with miR-125a OE, let-7e OE, or the control vector CT (B) or with miR-125a-5p sponge, let-7e-5p sponge, or the control vector CT (C) were stimulated for 6 h with LPS and then subjected to RIP assay using anti-Ago2 or IgG control Abs. TLR4 transcript levels were assayed in triplicate by Q-PCR (mean ± SEM; n = 6) and expressed as normalized fold enrichment in Ago2 IP (black columns) and leftover (white columns). (D–E) Surface molecule protein levels were measured by flow cytometry in THP-1 cells transduced with over-expressing or sponge vectors (gray bars on the left and right panels, respectively) for miR-125a (D) or let-7e (E) or their corresponding control vectors (black bars). Isotype control staining are shown in dotted histograms. Results from one representative experiment of 3 performed are shown. (* < 0.05; *** < 0.001).