The TNFAIP2 3′-UTR mutation confers translation repression in a miRNA dependent fashion. (A) The sequence of the 3′-UTR of TNFAIP2 (14:102673002-102673041) flanking the A→G mutation at 14:102673033 is shown, along with predicted binding sites for miR-223 and miR-181b. (B) The region of the TNFAIP2 3′-UTR shown in panel A containing germline (TNFAIP2 germ) or mutated sequence (TNFAIP2 mut) was cloned downstream of Renilla luciferase (psi-check2 vector) and transiently transfected into K562, 293T, and HS-5 cells. Expression of Renilla luciferase relative to firefly luciferase (an internal control present in the psi-check2 vector) after normalization to the germline TNAIP2 3′-UTR signal is shown. (C) Expression of miR-223 and miR-181b relative to RNU48 is shown. (D) 293T or K562 cells were cotransfected with the TNFAIP2 3′-UTR Renilla luciferase reporter constructs and a vector directing high level expression of either miR-223 or miR-181b. Expression of Renilla luciferase relative to firefly luciferase is shown. (E) Representative Western blot showing DICER1 protein expression in parent KLE cells (Wt) or KLE cells expressing shRNA directed against the red fluorescent protein (shRFP) or DICER1 (shDcrA). The upper band is nonspecific. GAPDH was used as a loading control. (F) The shRFP and shDicer KLE cells lines were transiently transfected with the TNFAIP2 3′-UTR Renilla luciferase reporter constructs. Expression of Renilla luciferase relative to firefly luciferase is shown. Data represent the mean ± SEM of 3-5 independent experiments.