Figure 4.
Negative regulation of miR-29a/b/c by BCR-induced MYC. (A) Intracellular staining for MYC in CXCR4/CD5 sorted subpopulations from 13 CLL samples. Results are represented as the ratio of MYC protein expression to the isotype control. The statistical differences were tested by paired t test. (Bi) Representative immunoblot of TRAF4 and MYC protein levels in primary CLL cells with BCR signaling activated by bead bound anti-IgM for indicated period of time. pAKT (phosphorylated AKT) and pERK (phosphorylated ERK) serve as positive controls for BCR pathway activation. (Bii) Densitometric quantification of TRAF4 levels for replicates of the experiment (n = 6) described in panel Bi. The statistical differences were tested by paired t test. (C-D) Analyses of miR-29a/b/c (C) and TRAF4 mRNA levels (D) in primary CLL cells (n = 6) with BCR signaling activated by bead-bound anti-IgM for 72 hours as described in panel B. For MYC induction by bead-bound anti-IgM, see panel Bi. The bead-bound anti-IgM did not affect CLL cell viability (supplemental Figure 7A). The statistical differences were tested by Wilcoxon matched pairs test. (E-F) Relationship between expression of miR-29a/b/c, MYC, or TRAF4 and the relative sensitivity to BCR ligation. CLL samples (n = 29) were treated with soluble anti-IgM and monitored for BCR-induced calcium flux by flow cytometry, which allowed us to stratify cases as relatively responsive to BCR ligation (n = 15) or unresponsive to treatment with soluble anti-IgM (n = 14; see supplemental Methods). The associations between BCR responsiveness and miR-29a/b/c (E) and TRAF4 or MYC (F) expression were plotted. Below each panel is a − or + in the row labeled BCR sig: on the left to indicate samples that were low responders or high responders, respectively. P value was tested by Mann-Whitney test. (G-H) Expression of miR-29a/b/c (G) or TRAF4 (H) in CLL patients (n = 96) divided according to MYC expression. All samples with available data on MYC mRNA levels were used in the analysis, and samples were stratified according to the terciles of MYC levels (low vs intermediate [interm] vs high; for distribution of MYC expression, see supplemental Figure 7B). The difference was tested by Mann-Whitney test. (I) Differences in miR-29 levels in B cells from transgenic iMyccα mice (MYC transgene controlled by the Ig heavy-chain enhancer, n = 3) compared with wildtype (WT) mice (n = 3). The differences were tested by unpaired t test. The error bars indicate SEM.

Negative regulation of miR-29a/b/c by BCR-induced MYC. (A) Intracellular staining for MYC in CXCR4/CD5 sorted subpopulations from 13 CLL samples. Results are represented as the ratio of MYC protein expression to the isotype control. The statistical differences were tested by paired t test. (Bi) Representative immunoblot of TRAF4 and MYC protein levels in primary CLL cells with BCR signaling activated by bead bound anti-IgM for indicated period of time. pAKT (phosphorylated AKT) and pERK (phosphorylated ERK) serve as positive controls for BCR pathway activation. (Bii) Densitometric quantification of TRAF4 levels for replicates of the experiment (n = 6) described in panel Bi. The statistical differences were tested by paired t test. (C-D) Analyses of miR-29a/b/c (C) and TRAF4 mRNA levels (D) in primary CLL cells (n = 6) with BCR signaling activated by bead-bound anti-IgM for 72 hours as described in panel B. For MYC induction by bead-bound anti-IgM, see panel Bi. The bead-bound anti-IgM did not affect CLL cell viability (supplemental Figure 7A). The statistical differences were tested by Wilcoxon matched pairs test. (E-F) Relationship between expression of miR-29a/b/c, MYC, or TRAF4 and the relative sensitivity to BCR ligation. CLL samples (n = 29) were treated with soluble anti-IgM and monitored for BCR-induced calcium flux by flow cytometry, which allowed us to stratify cases as relatively responsive to BCR ligation (n = 15) or unresponsive to treatment with soluble anti-IgM (n = 14; see supplemental Methods). The associations between BCR responsiveness and miR-29a/b/c (E) and TRAF4 or MYC (F) expression were plotted. Below each panel is a − or + in the row labeled BCR sig: on the left to indicate samples that were low responders or high responders, respectively. P value was tested by Mann-Whitney test. (G-H) Expression of miR-29a/b/c (G) or TRAF4 (H) in CLL patients (n = 96) divided according to MYC expression. All samples with available data on MYC mRNA levels were used in the analysis, and samples were stratified according to the terciles of MYC levels (low vs intermediate [interm] vs high; for distribution of MYC expression, see supplemental Figure 7B). The difference was tested by Mann-Whitney test. (I) Differences in miR-29 levels in B cells from transgenic iMyccα mice (MYC transgene controlled by the Ig heavy-chain enhancer, n = 3) compared with wildtype (WT) mice (n = 3). The differences were tested by unpaired t test. The error bars indicate SEM.

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