TNIP1 is the direct functional target of miR-517a/c. (A) Western blot analysis of lysates from HEK293 cells transfected with the indicated miRNAs. GAPDH was the loading control. (B and C) qPCR analysis of IL8 and TNF expression in HEK293 cells (B) transfected with two independent siRNAs against TNIP1 or (C) co-transfected with the indicated miRNAs and plasmid cDNAs. TNIP1 cDNA construct contained the complete coding sequence with a truncated 3'UTR. (D) Genome browser view of miR-517a/c binding site (red box) in TNIP1 3'UTR showing relative conservation across species. Also indicated is the presence of SNP rs72558377 in the binding site (red arrow). (E) Predicted base pairing between miR-517a and the TNIP1 binding site containing the major (C) and minor (T) alleles of rs72558377. '|' and ':' indicate Watson-Crick and wobble base pairing, respectively.(D) Both SNP alleles of the miR-517a/c target site in TNIP1 were cloned into the 3'UTR of a firefly luciferase reporter. The reporter also expressed Renilla luciferase from a separate promoter. The reporter was co-transfected with the indicated miRNAs in HEK293 cells. Firefly luciferase activity was normalized to that of Renilla luciferase. Statistical comparisons are relative to negative control (miR-neg). All values are means ± SD. B and C, n = 3. E, n = 4. *P ≤0.05, **P 0.01. miRNAs, micro RNAs; siRNAs, small interfering RNAs; SD, standard deviation; TNIP1, tumor necrosis factor alpha-induced protein 3 interacting protein 1.