Journal: Nature communications

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

doi: 10.1038/ncomms4000

Figure Lengend Snippet: (a) The schema shows naturally occurring d-flow (lesser curvature, LC) and stable flow ( s-flow) regions (greater curvature, GC) in the aortic arch. Also shown is the surgically induced d-flow in partial carotid ligation model in which three of the four caudal branches of the left common carotid artery (LCA) are ligated, while the contralateral right common carotid artery (RCA) remains untouched as an internal control. (b) Endothelial-enriched total RNAs obtained from intima of mouse (C57BL/6) left carotid (flow-disturbed LCA) and right carotid (contralateral control, RCA) at 48 h post-ligation, were analyzed by gene array (Illumina BeadChip). Hierarchical clustering analyses of mechanosensitive miRNAs found in LCA endothelium compared to that of RCA are shown as heat maps. The color represents the expression level of the gene. Red represents high expression, while green represents low expression. The expression levels are continuously mapped on the color scale provided at the top of the figure. Each column represents a single sample pooled from 3 different LCAs or RCAs, and each row represents a single miRNA probe (n=3). (c–f) Validation of miRNA microarray results by qPCR Quantitative PCR (qPCR), using additional independent RNA samples, was used to validate the above miRNA array data. Ten miRNAs (5 up-, 5 down-regulated miRNAs at 48 hours post-ligation) were selected based on fold-change by flow. The qPCR study validated the microarray results for (c) 5 up-regulated (miR-712, -330*, -699, -223, and 770–5p) and (d) 5 down-regulated (miR-195, -30c, -29b, -26b and let-7d) miRNAs at the 48 h time point (n=5 each, data shown as mean ± s.e.m; * p<0.05 as determined by paired t- test). To further validate whether the mechanosensitive genes that were identified in vivo responded specifically to shear stress, we tested expression of these miRNAs in vitro using immortalized mouse aortic endothelial cells (iMAECs) that were subjected to laminar (LS) or oscillatory stress (OS), mimicking s-flow and d-flow in vivo , respectively . Among the 9 different miRNAs examined, 7 miRNAs were differentially expressed under oscillatory shear (n=6 each, data shown as mean ± s.e.m; * p<0.05 as determined by paired t- test) (e) . These results showed that miR-712 was the most consistently and robustly upregulated miRNA both in vivo and under flow conditions in vitro.

Article Snippet: Intimal RNAs from three LCAs or RCAs were pooled to obtain ~30 ng total RNA . miRNA microarray was performed using Illumina Mouse v2 MicroRNA expression BeadChip array (Illumina) at the Emory Biomarker Service Center .

Techniques: Ligation, Control, Expressing, Biomarker Discovery, Microarray, Real-time Polymerase Chain Reaction, In Vivo, Shear, In Vitro

Journal: Nature communications

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

doi: 10.1038/ncomms4000

Figure Lengend Snippet: (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 (0–48h) (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 hours as above in (b) 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 immortalized mouse aortic endothelial cells (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 obtained from control C57Bl6 mice were subjected to fluorescence in situ hybridization using digoxigenin-labeled miR-712 probe and anti-digoxigenin antibody, which was detected by tyramide signal amplification method using Cy-3 and confocal microscopy (shown in red), (n=6). Blue: DAPI nuclear stain; Green: auto-fluorescent elastic lamina; Arrows indicate cytosolic miR-712 expression. White scale bars = 20 μm. (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 2 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-day post ligation) were 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).

Article Snippet: Intimal RNAs from three LCAs or RCAs were pooled to obtain ~30 ng total RNA . miRNA microarray was performed using Illumina Mouse v2 MicroRNA expression BeadChip array (Illumina) at the Emory Biomarker Service Center .

Techniques: Expressing, Ligation, Shear, Control, Fluorescence, In Situ Hybridization, Labeling, Amplification, Confocal Microscopy, Staining