MicroRNAs (miRs) are involved in the initiation, progression and dissemination of CLL cells (Calin GA, Croce CM. Blood 114:4761, 2009). Recent studies showed that high levels of miR-155, previously shown to regulate hematopoietic cell development, are expressed in CLL cells. Because transgenic miR-155 overexpression in the mouse stimulates B-cell proliferation, it is thought that miR-155 plays a role in the pathogenesis of CLL (Calin GA et al. N Engl J Med 353:1793, 2005). STAT3 is constitutively activated in CLL and induces the transcription of several STAT3-regulated genes. A recent study demonstrated that STAT3 activates miR-21 and miR-181b-1 (Iliopolus D. et al. Mol Cell 39:493, 2010). Therefor we wondered whether STAT3 enhances the expression of miR-155 in CLL cells. Because a sequence analysis revealed that the promoter of miRNA-155 harbors γ-interferon activation sequence-like elements typically activated by STAT3, we sought to determine whether STAT3 directly activates miR-155 expression. We generated truncated constructs of the miR-155 promoter, co-transfected them into MM1 cells together with STAT3 small interfering (si) RNA (siRNA), and assessed their luciferase activity. The luciferase activity data suggested that of the two putative STAT3 binding sites only one site is involved in STAT3 induced transcription because STATR3-siRNA reduced the activity of miRNA-155 promoter of constructs that harbor this site. To confirm these data we performed an electrophoretic mobility shift assay (EMSA) and chromatin immune-precipitation (ChIP). The EMSA confirmed that STAT3 bound the miR-155 promoter in fresh CLL cells, and ChIP confirmed that STAT3 bound one putative STAT3-binding site in the miR-155 promoter but not to the other, as demonstrated by the luciferase assay; STAT3 co-immuno-precipitated only one putative STAT3 binding region of miR-155 promoter and other STAT3-regulated genes. Finally, STAT3-small hairpin RNA (shRNA) downregulated miR-155 and other STAT3-regulated genes, suggesting that constitutively activated STAT3, binds miR-155 promoter and induces miR-155 transcription in CLL cells.
Keating:Celgene Corporation: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Xcenda: Consultancy, Speakers Bureau.
Asterisk with author names denotes non-ASH members.