Figure 5: Anti-miR-712 silences miR-712 and restores TIMP3 expression in vivo. | Nature Communications

Figure 5: Anti-miR-712 silences miR-712 and restores TIMP3 expression in vivo.

From: The atypical mechanosensitive microRNA-712 derived from pre-ribosomal RNA induces endothelial inflammation and atherosclerosis

Figure 5

(a) TexRed-615-labelled control anti-miR or saline was injected (s.c.) in C57Bl6 mice. Carotid arteries were dissected out 24 h later, and using Zeiss 510 confocal microscope z-stack images of the en face sections were obtained. Images were rendered to three dimensions, bounding box was drawn on the area of interest and scaled coordinate axes were drawn and fluorescence signals from Red channel were processed and quantified. Graph shows mean fluorescence intensity of TexRed-615 signals from carotids (n=5, data shown as mean±s.e.m.). (b) Following partial carotid ligation and HFD for 2 weeks, ApoE−/− mice were injected with 64Cu-labelled anti-miR-712 via tail vein. After 3 h, aortic trees including carotids were prepared and autoradiographed, which was used to quantitate percentage of injected dose per gram (n=6, data shown as mean±s.e.m.; *P<0.05 as determined by Student’s t-test). Scale bar, 1 mm. (c) To determine the optimal dose of anti-miR-712, C57Bl6 mice were injected with anti-miR-712 daily for 2 days (s.c. at 5, 20 or 40 mg kg−1 per day) and followed by partial carotid ligation. Mismatched anti-miR-712 (40 mg kg−1 per day) was used as a control. Endothelial-enriched RNAs were prepared from LCA and RCA obtained at 4 days post ligation, and miR-712 expression was determined by qPCR showing optimal effect at 5 mg kg−1 dose (n=4 each, data shown as mean±s.e.m.; *P<0.05 as determined by paired t-test). (d–f) ApoE−/− mice were partially ligated and fed HFD for 1 week (d,e) or 2 weeks (f). RCA and LCA frozen sections obtained from these mice were used for immunofluorescence staining with antibody specific to TIMP3 shown in red (scale bar, 20 μm) (d) or versican fragment peptide DPEAAE shown in red (scale bar, 20 μm) (f), and in situ zymography using DQ-gelatin (green) to determine MMP activity (e). As a control for the MMP activity assay, some LCA and RCA sections were incubated with MMP inhibitor 1. Blue: DAPI and green: autofluorescent elastic lamina (L=lumen of the artery). Scale bar, 50 μm.

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