. 2013:4:3000.

doi: 10.1038/ncomms4000.

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

Dong Ju Son¹, Sandeep Kumar¹, Wakako Takabe², Chan Woo Kim³, Chih-Wen Ni⁴, Noah Alberts-Grill⁵, In-Hwan Jang⁴, Sangok Kim⁶, Wankyu Kim⁶, Sang Won Kang⁶, Andrew H Baker⁷, Jai Woong Seo⁸, Katherine W Ferrara⁸, Hanjoong Jo³

Affiliations

¹ 1] Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322, USA [2] Division of Cardiology, Department of Medicine, Emory University, Atlanta, Georgia 30322, USA [3].
² 1] Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322, USA [2] Division of Cardiology, Department of Medicine, Emory University, Atlanta, Georgia 30322, USA.
³ 1] Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322, USA [2] Division of Cardiology, Department of Medicine, Emory University, Atlanta, Georgia 30322, USA [3] Department of Bioinspired Science, Ewha Womans University, Seoul 120-750, South Korea.
⁴ Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322, USA.
⁵ Division of Cardiology, Department of Medicine, Emory University, Atlanta, Georgia 30322, USA.
⁶ Department of Life Science, Ewha Womans University, Seoul, South Korea.
⁷ Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow G12 8TA, UK.
⁸ Department of Biomedical Engineering, University of California, Davis, California 95616, USA.

PMID: 24346612
PMCID: PMC3923891
DOI: 10.1038/ncomms4000

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

Dong Ju Son et al. Nat Commun. 2013.

. 2013:4:3000.

doi: 10.1038/ncomms4000.

Authors

Affiliations

¹ 1] Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322, USA [2] Division of Cardiology, Department of Medicine, Emory University, Atlanta, Georgia 30322, USA [3].
² 1] Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322, USA [2] Division of Cardiology, Department of Medicine, Emory University, Atlanta, Georgia 30322, USA.
³ 1] Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322, USA [2] Division of Cardiology, Department of Medicine, Emory University, Atlanta, Georgia 30322, USA [3] Department of Bioinspired Science, Ewha Womans University, Seoul 120-750, South Korea.
⁴ Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322, USA.
⁵ Division of Cardiology, Department of Medicine, Emory University, Atlanta, Georgia 30322, USA.
⁶ Department of Life Science, Ewha Womans University, Seoul, South Korea.
⁷ Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow G12 8TA, UK.
⁸ Department of Biomedical Engineering, University of California, Davis, California 95616, USA.

PMID: 24346612
PMCID: PMC3923891
DOI: 10.1038/ncomms4000

Abstract

MicroRNAs (miRNAs) regulate cardiovascular biology and disease, but the role of flow-sensitive microRNAs in atherosclerosis is still unclear. Here we identify miRNA-712 (miR-712) as a mechanosensitive miRNA upregulated by disturbed flow (d-flow) in endothelial cells, in vitro and in vivo. We also show that miR-712 is derived from an unexpected source, pre-ribosomal RNA, in an exoribonuclease-dependent but DiGeorge syndrome critical region 8 (DGCR8)-independent manner, suggesting that it is an atypical miRNA. Mechanistically, d-flow-induced miR-712 downregulates tissue inhibitor of metalloproteinase 3 (TIMP3) expression, which in turn activates the downstream matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs) and stimulate pro-atherogenic responses, endothelial inflammation and permeability. Furthermore, silencing miR-712 by anti-miR-712 rescues TIMP3 expression and prevents atherosclerosis in murine models of atherosclerosis. Finally, we report that human miR-205 shares the same 'seed sequence' as murine-specific miR-712 and also targets TIMP3 in a flow-dependent manner. Targeting these mechanosensitive 'athero-miRs' may provide a new treatment paradigm in atherosclerosis.

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Conflict of interest statement

Conflict of Interest

None.

Figures

Figure 1. miR-712 is a flow-sensitive microRNA upregulated by *d-flow in vitro* and *in vivo*
(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.*

**Figure 2. miR-712 is atypically derived from RN45S gene in XRN-1-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 (0–48h) (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).

**Figure 3. TIMP3 is the direct target of miR-712**
(a) shows TIMP3 as a potential target of miR-712 and its link to putative downstream metalloproteinase targets. (b) shows the seed sequence of miR-712 and complementary 3′-UTR sequence of TIMP3. (c) iMAECs transfected with dual-luciferase reporter plasmids containing wild-type (WT) or mutant TIMP3-3′UTR, were treated with pre-miR-712 or control pre-miR. Firefly luciferase activity (normalized to control *Renilla* luciferase) indicating TIMP3 expression was determined using Luc-Pair miR Luciferase Assay Kit (n=3 each, data shown as mean ± s.e.m; * p<0.05 as determined by Student’s t-test). (d) *TIMP3* expression in iMAECs determined by qPCR was decreased by exposure to OS compared to LS or ST for 24 h (n=6, data shown as mean ± s.e.m; * p<0.05 as determined by Student’s t-test). (e–g) Representative Western blots show modulation of TIMP3 expression by treatment with shear stress in the presence of pre-miR-712 or anti-miR-712 for 24 h in iMAECs. (e) TIMP3 expression decreased by OS compared to LS for 24 h. (f) Treatment with pre-miR-712 (20 nM) down-regulated TIMP3 expression under LS condition. (g) anti-miR-712 (400 nM) treatment rescued OS-induced loss of TIMP3. β-actin was used as internal loading control. Full sized scans of all Western blots are provided in Supplementary Figure S22.

**Figure 4. miR-712 induces endothelial inflammation and hyperpermeability changes**
(a) iMAECs were exposed to static, LS, or OS for 24 h, and the conditioned media were used for TNFα ELISA. LPS was used as a positive control. (b) iMAECs were transfected with TIMP3 expression vector (TIMP3-OE), TIMP3 siRNA (150nM), pre-miR-712 (20nM), anti-miR-712 (400nM), and respective controls for 24 h. Cells were then exposed to static, LS or OS for 24 h, and s-TNFα in conditioned media was determined by ELISA. (n=6, data shown as mean ± s.e.m; * p<0.05 as determined by 1-way ANOVA). (c) iMAECs were transfected with pre-miR-712 (20nM) or control and leukocyte adhesion assay was performed using 2.5 X 10⁵ fluorescent-labeled J774.4 mouse monocyte cells. Adherent cells were counted (n=5 independent experiments, data shown as mean ± s.e.m; * p<0.05 as determined by 1-way ANOVA). **(d)** iMAECs were transfected with TIMP3-OE or its GFP vector control (GFP-OE), TIMP3 or scrambled control siRNA (siTIMP3 or siScr, 150nM), pre-miR-712 or control-pre-miR (20nM), pre-miR-712 with GFP-OE or TIMP3-OE, and no treatment (control), transfection (mock) or no cell controls for 24 h. Cells were then exposed to static, LS or OS for 24 h, and endothelial permeability was determined using a FITC-dextran based *in vitro* vascular permeability kit (Cultrex). Fluorescence signals from FITC-dextran in the lower chamber were quantified and plotted as arbitrary fluorescence units (n=3 each from two independent experiments, data shown as mean ± s.e.m; * p<0.05 as determined by 1-way ANOVA).

Figure 5. Anti-miR-712 silences miR-712 and restores TIMP3 expression *in vivo*
(a) TexRed-615-labeled 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 3D, bounding box was drawn on the area of interest and scaled cordinate 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 high-fat diet for 2 weeks, ApoE^−/− mice were injected with ⁶⁴Cu-labelled anti-miR-712 via tail-vein. After 3 hours, aortic trees including carotids were prepared and autoradiographed, which was used to quantitate percentage of injected dose per gram (%ID/g) (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/day dose) and followed by partial carotid ligation. Mismatched anti-miR-712 (40 mg/kg/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 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 high-fat diet for 1 week (d and 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 =20μm. (d) or versican fragment peptide DPEAAE shown in red. Scale bar =20μm. (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 =50μm.

**Figure 6. Anti-miR-712 or adenoviral TIMP3 reduces atherosclerosis in ApoE^−/− mice**
For acute study, *ApoE*⁻^/⁻ mice were pretreated twice with anti-miR-712 or mismatched control (5mg/kg each, *s.c.*) or saline on 1 and 2 days prior to partial ligation. Mice were then fed a high-fat diet and anti-miR and control treatments were continued (twice a week *s.c.*) for two weeks. (a) Aortic trees including the carotids were dissected and examined by bright field imaging and lesion area was quantified in (d) n=10 each, data shown as mean ± s.e.m; * p<0.05 as determined by 1-way ANOVA). Scale bar =1mm. (b) Frozen sections prepared from the middle parts of these arteries, noted by red arrows in (a) were stained with Oil-Red-O and plaque size was quantified in (e) (n=10 each, data shown as mean ± s.e.m; * p<0.05 as determined by 1-way ANOVA). Scale bar =200μm. (c) Representative confocal imaging of frozen sections immunostained with CD45 antibody is shown (n=10). Scale bar =20μm. For chronic study, *ApoE*⁻^/⁻ mice were fed western-diet (without any partial ligation surgery) and were treated with anti-miR-712 (5 mg/kg, twice a week, *s.c.*) or mismatched control for 3 months (n=10 each). (f) Aortic trees were dissected and examined by *en face* Oil-Red-O staining and the lesion area was quantified in (h) n=4–6 in each group, data shown as mean ± s.e.m; * p<0.05 as determined by Student’s t-test). Scale bar =2mm. Some aortic arches were longitudinally sectioned and stained with Oil-Red-O (g) and plaque size was quantified in (i) (n=4 each, data shown as mean ± s.e.m; * p<0.05 as determined by Student’s t-test). Scale bar =2mm. (j) For TIMP3 overexpression, *ApoE*⁻^/⁻ mice were injected once with AdTIMP3 (10⁸ pfu/animal, via tail vein) or control virus (RAD60, 10⁸ pfu/animal) 5 days before partial carotid ligation and high fat diet for 2 weeks. (j) Aortic trees including the carotids were dissected and examined by bright field imaging and lesion area was quantified in (k) (n=5, data shown as mean ± s.e.m; * p<0.05 as determined by Student’s t-test). Scale bar =1mm.

**Figure 7. miR-205 is a flow-sensitive human homolog of murine miR-712**
**(a)** The highlighted region in yellow shows the seed sequence match between the murine miR-712 and murine and human miR-205 and those shown in blue indicate additional conserved sequences. **(b)** The putative targets of miR-712 and miR-205 obtained from TargetScan were compared. Venn diagram depicts the common gene targets of miR-205 and miR-712. **(c)** Expression of precursor- and mature miR-205 in iMAECs exposed to static, LS or OS for 24 h (n=4; data shown as means ± s.e.m; *, p<0.05 as determined by Student’s t-test). **(d)** Expression of precursor- and mature miR-205 in human aortic endothelial cells (HAECs) exposed to static, LS or OS for 24 h (n=4; data shown as means ± s.e.m; *, p<0.05 as determined by Student’s t-test). **(e)** Expression of mature-miR-205 was determined using the RNAs obtained from the endothelial-enriched (intimal region) and the leftover medial and adventitia region (M +A) of the LCA and RCA at 24 h and 48 h post-partial ligation, respectively. Expression of *TIMP3* was determined by qPCR in (f) iMAECs and **(g)** HAECs transfected with increasing concentrations of pre-miR-205 or control-pre-miR compared to vehicle controls (mock) (n=3; data shown as means ± s.e.m; * *, p<0.05 as determined by Student’s t-test).

**Figure 8. Summary and working hypothesis**
miR-712 induces inflammation and atherosclerosis by targeting TIMP3. *D-flow* stimulates miR-712 expression in endothelium by an XRN1-dependent mechanism. Increased miR-712 stimulates endothelial inflammation, permeability and ECM fragmentation by downregulating TIMP3, which is a critical inhibitor of matrix metalloproteinases (MMPs and ADAMs). Decreased TIMP3 expression by miR-712 induces inflammation and atherosclerosis by activating a multitude of metalloproteinases: (1) ADAM17/TACE which releases soluble-TNFα that may induce local and systemic inflammation; (2) ADAMs that shed junctional VE-cadherin, increasing permeability that facilitates LDL and leukocyte infiltration; (3) ADAMTS leading to versican fragmentation; (4) MMPs leading to ECM degradation leading to vessel wall remodeling. Additionally, miR-712 expression is also increased in whole blood and vascular smooth muscle cells (VSMCs) suggesting either transfer of miR-712 from endothelium to these compartments or increase its local production in these compartments. Increased miR-712 in VSMCs induces their migration while circulating miR-712 may affect blood leukocytes further contributing to atherogenesis.

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

Affiliations

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

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

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Associated data

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