Fig. 3.

miR-652-3p negatively regulates the endothelial repair gene Ccnd2.

(a) Bioinformatics algorithms predicted two miR-652 binding spots in the 3′UTR of human CCND2 and one binding spot in the CDS of mouse Ccnd2. (b) Intimal EC expression of Ccnd2 mRNA at 28 days post-injury. (c) The effect of miR-652-3p on luciferase activity of psiCHECK-2 Ccnd2 promoter constructs. Empty psiCHECK-2 (psiCHECK), WT Ccnd2 CDS (wild-type murine Ccnd2), and Ccnd2 CDSmut (mutated murine Ccnd2) constructs were co-transfected with scrambled pre-miRNA (30 nM) or synthetic miR-652-3p (0 nM, 15 nM, or 30 nM). (d) Western blot analysis of CCND2 expression in HUVECs. Band intensities were normalized with respect to β-actin band intensity. *P < .05 vs. Ctrl, †P < .05 vs. LNA ctrl. (e-g) Flow cytometric HUVEC proliferation assays under shear flow conditions. (e) HUVECs were treated with LNA-652-5p and/or LNA-652-3p, or siCCND2 (CCND2-specific siRNA). (f) HUVECs were treated with 652-5p mimic and/or 652-3p mimic (miR-652-5p mimic and miR-652-3p mimic, respectively), with (+) or without (−) overexpression of CCND2. (g) HUVECs, with (CCND2^+5pMRE) or lacking (CCND2^Δ5pMRE) the miR-652-3p recognition element, were treated by a miR-652-3p mimic. (h-l) Carotid-injured Mir652^−/− mice were perivascularly administered a single 4-nmol dose of scrambled control or a Ccnd2-specific siRNA on days 14 and 21 post-carotid injury. On day 28 post-injury, mice were sacrificed, and intimal EC expression of (h) Ccnd2 mRNA and (i) Ccnd2 protein, (j) plaque area, as well as (k) endothelial recovery and (l) proliferation were analyzed. Scale bar = 100 μm. n = 12–18 mice per experimental group. Data reported as means ± SEMs. *P < .05 vs. first group, †P < .05 vs. second group.