Experimental validation of the interaction of miR-9 and miR-30, which are expressed highly in GC B cells compared with plasma cells and target the transcription factor PRDM1. (A) Base-pairing of the 3′UTR of PRDM1 gene with the 5′ seed region of miR-9 and the miR-30 family. The miR-30 regions include 3 sites complementary to nucleotides 2-8 (UTR position 408), nucleotides 1-8 (UTR position 2370), and nucleotides 2-8 (UTR position 2383) on the miRNA, respectively. The miR-9 regions include 3 sites complementary to nucleotides 1-7 (UTR position 1459), nucleotides 2-8 (UTR position 2108), and nucleotides 2-8 (UTR position 2323) on the miRNA, respectively. These sites are highly conserved across several species, with the exception of one miR-9 site (UTR position 1459) that is present only in humans. (B) Effects of overexpression of miR-9 and 2 members of the miR-30 family, miR-30b and miR-30d, in plasma cell myeloma-derived U266 cells in 3 separate experiments. Expression of PRDM1 was measured 24 hours after transfection with a scrambled control with no complementarity to the human genome, a hairpin precursor for miR-30b, a hairpin precursor for miR-30d, or a hairpin precursor for miR-9. (C) Relative PRDM1 protein expression from a representative experiment (from 3 replicates) transfecting a scrambled control versus a precursor for miR-9, miR-30b, and miR-30d in U266 cells. (D) Average expression of PRDM1 relative to actin over 3 Western blots of U266 cells transfected with a scrambled control versus a precursor for miR-9, miR-30b, and miR-30d. P < .05 for miR-30b and miR-30d; P = .08 for miR-9. (E) A luciferase-expressing vector was coupled to the 3′UTR of PRDM1. Repression of luciferase activity was observed upon overexpression of miR-30b, miR-30d, and miR-9 (P < .05 in all 3 cases) but rescued to the activity level of the empty vector control (P > .5 in all 3 cases) when the seed sequence of the miRNAs was mutated. Displayed is the average of 3 separate experiments.