Inhibition of miR-21 can abrogate the effects of TGF-β on hematopoietic cells. miR-21 levels were measured in leukemic K562 cells after 48-hour treatment with LNA inhibitor of miR-21 and compared with mismatched control (mm control). miR-21 transcripts were significantly decreased after LNA treatment (50 mM) as measured by quantitative RT-PCR (mean ± SEM of 2 experiments, Student’s t-test, P < .001). (B) SMAD7 protein expression is increased after miR-21 inhibition (50 mM, 48-hour treatment) in HL-60, TF-1 cell lines and in leukocytes from 2 MDS patients, as shown by immunoblotting. Actin was used as a protein-loading control. (C) miR-21 inhibitor treatment leads to decreased p-SMAD2 in MDS leukocytes when compared with mismatched-control LNAs (50 mM). (D) Primary CD34+ progenitors were nucleofected with GFP coexpressing anti-SMAD7 (or scrambled control) shRNA constructs18 and sorted after 24 hours. GFP-positive cells were grown in methylcellulose in the presence and absence of TGF-β (20 ng/mL) and miR-21 inhibitor (50 mM), and erythroid colonies were counted after 14 days. miR-21 inhibitor treatment abrogates the inhibitory effects of TGF-β on erythroid colonies (mean ± SEM of 2 independent experiments, Student’s t-test, P = .04) in cells that were transfected with scrambled shRNA controls. SMAD7 shRNA–transfected progenitors did not demonstrate any reversal of TGF-β–induced inhibition after miR-21 inhibitor treatment. (E) miR-21 and GFP were co-overexpressed in leukemic K562 cells using the pMIF-cGFP-Zeo vector, leading to decreased levels of SMAD7 as detected by immunoblotting when compared with the GFP-only control. (F) Primary CD34+ progenitors were nucleofected with miR-21and GFP or with the GFP-only control and sorted after 24 hours. GFP-positive cells were grown in methylcellulose in the presence of TGF-β (20 ng/mL) and hematopoietic colonies were counted after 14 days. (G) miR-21 overexpression led to decreased erythroid colony formation (mean ± SEM of 2 independent experiments, Student’s t-test, P = .01) and also led to qualitative reductions in colony sizes.
