FIGURE 2.

miR-21 regulates multiple members of DOCK family proteins. A, PASMCs were treated with 3 nm BMP4 (24 or 48 h) or transfected with control mimic (cel-miR-67) or miR-21 mimic. Total cell lysates were subjected to immunoblotting with anti-DOCK7 or anti-GAPDH (loading control) antibody. B, all members of the DOCK family except DOCK3 and DOCK8 were enriched 3–34-fold upon bio-miR-21 pulldown in comparison with control (bio-cel-miR-67) pulldown. C, total RNA was harvested from PASMCs transfected with control or anti-miR-21 oligonucleotides, and mRNA levels of the indicated DOCK genes relative to GAPDH were measured by qRT-PCR. Data represent fold changes of the mRNA levels in anti-miR-21-transfected cells in comparison with control oligonucleotide-transfected cells ±S.E. from triplicates. *, p < 0.01. D, COS7 cells were transfected with a luciferase reporter containing 3′-UTR of DOCK1, -2, -4, -5, -6, -7, -9, -10, or -11 gene transcript and cel-miR-67 (control) or miR-21 mimic. Relative luciferase activities were examined. The miR-21 sensor and an empty luciferase vector (Vector) were used as controls. Data represent fold changes in the luciferase activity in miR-21-mimic-transfected cells compared with control-mimic-transfected cells ±S.E. from triplicates. *, p < 0.01. E, PASMCs were treated with 3 nm BMP4 for 24 h. mRNA levels of the indicated DOCK genes relative to GAPDH were measured by qRT-PCR. Data represent fold changes of the mRNA level in BMP4-treated cells compared with mock-treated cells ±S.E. from triplicates. *, p < 0.01.