miR-17∼92 directly targets the ITIM-containing CD22 and FCGR2B to stimulate the BCR response. (A) P493-6 cells grown in the absence (MYC^HIGH) or presence (MYC^LOW) of 1μM doxycycline were treated with control mimic or miR-17∼92 mimic mix for 48 hours. RNA was isolated and qPCR was performed to quantitate changes in the FCRL4, FCGR2B, CD22, and CD72 mRNAs. GAPDH was used as an endogenous control and values are relative to MYC high/CTRL mimic. The average of 4 repetitions is reported. Error bars represent the SD. *P < .05 and **P < .01 by Student t test. (B) Changes in FCGR2B and CD22 protein levels were examined by western blotting on cells from Figure 4A. GAPDH was used as a loading control. (C-D) Dual luciferase assays performed in HCT-116 Dicer hypomorph cells cotransfected with CD22 3′UTR or FCGR2B 3′UTR luciferase sensors (panels C and D, respectively) and either control or miRNA mimics. Schematic of predicted 3′UTR-miRNA interactions are depicted (top panel). Shaded nucleotides indicate regions mutated to disrupt the mRNA-miRNA interaction in the mut 3′UTR constructs. Changes in luciferase activity upon treatment with miRNA mimics are plotted (bottom panel). Values were normalized to luciferase activity from control mimic-transfected cells. Error bars represent SD of 3 independent experiments. *P < .05 and **P < .01 by Student t test. (E) P493-6 cells grown in the presence (MYC^LOW) of 1μM doxycycline and treated with control, anti-CD22, or anti-FCGR2B small interfering RNA (siRNA) for 48 hours prior to addition of 10 μg/mL soluble anti-IgM for 15 minutes to ligate the BCR. Western blotting was performed for CD22, FCGR2B, P-BLNK, total-BLNK, and actin.