Figure 2: miR-712 is atypically derived from RN45S gene in XRN1-dependent manner.

(a) Expression of miR-712 was determined by qPCR using endothelial-enriched RNA obtained from LCA and RCA following partial carotid ligation in C57Bl6 mouse (0–48 h) (n=4, data shown as mean±s.e.m.; *P<0.05 as determined by paired t-test). (b,c) Expression of pre-miR-712 and mature miR-712 by d-flow in LCA and RCA endothelium following partial ligation at 24 and 48 h as above in a was quantitated by miScript miRNA qPCR assay (n=8 each; *P<0.05 as determined by Student’s t-test). (d,e) Expression of pre-miR-712 and mature miR-712 was measured by miScript miRNA qPCR in iMAECs exposed to laminar (LS), oscillatory shear (OS) or static (ST) for 24 h (n=6, data shown as mean±s.e.m.; *P<0.05 as determined by Student’s t-test). (f) Aortic arch (LC and GC) and LCA and RCA obtained at 2 days post ligation from C57Bl6 mice were subjected to fluorescence in situ hybridization using DIG-labelled miR-712 probe and anti-DIG antibody, which was detected by tyramide signal amplification method using Cy3 and confocal microscopy (shown in red), (n=6). Blue: DAPI nuclear stain; green: autofluorescent elastic lamina; arrows indicate cytosolic miR-712 expression. White scale bars, 20 μm. Panel g shows the potential structure and processing of pre-ribosomal RNA gene, RN45s, which is composed of 18S, 5.8S and 28S rRNA sequences with two intervening spacers ITS1 and ITS2. The sequences matching murine miR-712 in ITS2 and its putative human counterpart miR-663 in ITS1 are indicated as well. (h–j) Expression of DICER, DGCR8 and XRN1 in mouse RCA and LCA (2 days post ligation) was determined by qPCR (n=4, data shown as mean±s.e.m.; *P<0.05 as determined by paired t-test). (k,l) XRN1 expression in iMAECs and HUVECs exposed to LS or OS for 24 h was determined by qPCR (n=3 each, data shown as mean±s.e.m.; *P<0.05 as determined by Student’s t-test). (m) miR-712 expression was induced by treating iMAECs with XRN1 siRNA but not by DGCR8 siRNA and DICER1 siRNA (n=3 each, data shown as mean±s.e.m.; *P<0.05 as determined by Student’s t-test).