Endothelial cells express miR-126. (A) (Upper) Total RNA was harvested from various mouse tissues and analyzed by Northern blotting for miR-126 RNA. (Lower) Ethidium bromide staining of total RNA. (B) (Upper) Total RNA was harvested from various cell types and analyzed by Northern blotting for miR-126 RNA. (Lower) Ethidium bromide staining of total RNA. (C) Total RNA was harvested from various cell types and analyzed by quantitative RT-PCR for miR-126 RNA. (D) HUVEC were transfected with AS oligonucleotides or premiR-126 oligonucleotides and then cultured for 3 days. TNF-α at 1 ng/ml was added for 16 h. Total RNA was harvested and analyzed by Northern blotting for miR-126 RNA levels (Upper) and by ethidium bromide staining (Lower).

miR-126 suppresses gene expression by targeting a miR-126-binding site. (A) miR-126 is partially complementary to a region in the VCAM-1 3′-UTR. (Upper) Schematic of VCAM-1 mRNA and miR-126. (Lower) The sequence of miR-126 and its potential matching site in the VCAM-1 3′-UTR. (B) The complementary miR-126-binding site was inserted downstream of a luciferase reporter on the pMIR-Report plasmid and transfected into HEK293 cells with varying concentrations of premiR-126 (n = 3 ± SD; *, P < 0.01 for 0 vs. 20 nM premiR-126). (C) The partially complementary miR-126-binding site found in the VCAM-1 3′-UTR (or a mutated binding site) was inserted downstream of a luciferase reporter on the pMIR-Report plasmid and transfected into HEK293 cells with varying concentrations of premiR-126 (n = 3 ± SD; *, P < 0.001 for 0 vs. 30 nM premiR-126).

miR-126 suppresses leukocyte adhesion ex vivo. (A) HUVEC were transfected with premiR-126 or a control oligonucleotide for 2 days and then treated with TNF-α or control for 6 h. BCECF-AM-labeled leukocytes were added to the HUVEC, incubated at 37°C for 45 min, and then washed. Adherent cells were photographed with a fluorescent camera. (B) HUVEC were transfected with premiR-126 or a control oligonucleotide for 2 days and then treated with TNF-α or control for 6 h. HRP-labeled leukocytes were added to HUVEC, incubated at 37°C for 45 min, and then washed. Adherent cells were lysed, and HRP was assayed by a spectrophotometer at A₄₅₀, and then calibrated to a standard curve (n = 2 ± SD; P = 0.1 for 0 vs. 30 nM premiR-126). (C) HUVEC were transfected with premiR-126 or a control oligonucleotide for 2 days and then treated with TNF-α or control for 6 h. Cell lysates were immunoblotted with antibody to VCAM-1. (D) HUVEC were transfected with AS-miR-126 or an AS-control oligonucleotide for 2 days and then treated with TNF-α or control for 6 h. HRP-labeled leukocytes were added to the HUVEC, incubated at 37°C for 45 min, and then washed. Adherent cells were lysed, and HRP was assayed by a spectrophotometer at A₄₅₀ and calibrated to a standard curve (n = 3 ± SD; *, P = 0.02 for 0 vs. 30 nM AS-miR-126). (E) HUVEC were transfected with AS-miR-126 or a control oligonucleotide for 2 days and then treated with TNF-α or control for 6 h. Cell lysates were immunoblotted with antibody to VCAM-1. (F) HUVEC were treated with TNF-α or control for 6 h. HUVEC were incubated with antibody to VCAM-1, ICAM-1, or control IgG for 30 min at 37°C. HRP-labeled leukocytes were then added to the HUVEC, incubated at 37°C for 45 min, and washed. Adherent cells were lysed, and HRP was assayed by a spectrophotometer at A₄₅₀ and calibrated to a standard curve (n = 3 ± SD; *, P < 0.02).

miR-126 suppresses VCAM-1 expression. (A) TNF-α induction of VCAM-1 expression in HUVEC. HUVEC were stimulated for 16 h with increasing doses of TNF-α, and cell lysates were immunoblotted with antibody to VCAM-1. (B) Endogenous miR-126 decreases VCAM-1 protein levels, knocking down miR-126. HUVEC were transfected with a control oligonucleotide (AS-control) or with an oligonucleotide antisense to miR-126 (AS-miR-126) to knock down endogenous miR-126 and then cultured for 3 days. Total protein was harvested and analyzed by immunoblotting for VCAM-1. (C) Endogenous miR-126 decreases VCAM-1 protein levels, increasing miR-126. HUVEC were transfected with a premiR-126 oligonucleotide to increase miR-126 and then cultured for 3 days. TNF-α at 1 ng/ml was added for 16 h. Total protein was harvested and analyzed by immunoblotting for VCAM-1. (D) HUVEC were transfected with 30 nM AS-miR-126 or premiR-126 or a control oligonucleotide and stimulated with TNF-α, and immunoblots were performed as above. The intensity of the signal was quantified and compared with the control transfected cells (n = 6 ± SD; *, P < 0.03 compared with control). (E) HUVEC were treated with a control oligonucleotide or premiR-126 and stimulated with TNF-α as above, and the expression of VCAM-1 was measured by FACS (n = 4 ± SD; *, P < 0.01). (F) miR-126 does not affect steady-state VCAM-1 mRNA levels. HUVEC were transfected with premiR-126 and treated with TNF-α for 3 h, and total RNA was analyzed by Northern blotting for VCAM-1. (G) miR-126 does not affect the stability of VCAM-1 mRNA. HUVEC were transfected with premiR-126, treated with TNF-α for 3 h, and then exposed to actinomycin D. RNA was isolated at various times after actinomycin D and analyzed by quantitative RT-PCR for VCAM-1 and GAPDH mRNA levels.