MicroRNA-126 regulates endothelial expression of vascular cell adhesion molecule 1

Abstract

Adhesion molecules expressed by activated endothelial cells play a key role in regulating leukocyte trafficking to sites of inflammation. Resting endothelial cells normally do not express adhesion molecules, but cytokines activate endothelial cells to express adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), which mediate leukocyte adherence to endothelial cells. We now show that endothelial cells express microRNA 126 (miR-126), which inhibits VCAM-1 expression. Transfection of endothelial cells with an oligonucleotide that decreases miR-126 permits an increase in TNF-alpha-stimulated VCAM-1 expression. Conversely, overexpression of the precursor to miR-126 increases miR-126 levels and decreases VCAM-1 expression. Additionally, decreasing endogenous miR-126 levels increases leukocyte adherence to endothelial cells. These data suggest that microRNA can regulate adhesion molecule expression and may provide additional control of vascular inflammation.

Fig. 1.

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).

Fig. 2.

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).

Fig. 3.

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.

Fig. 4.

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).