Accepted Manuscript

MicroRNA miR-320a modulates induction of HO-1, GCLM and OKL38 by oxidizedphospholipids in endothelial cells.

Waltraud Schrottmaier, Olga V. Oskolkova, Gernot Schabbauer, Taras Afonyushkin

PII: S0021-9150(14)00175-0

DOI: 10.1016/j.atherosclerosis.2014.03.026

Reference: ATH 13481

To appear in: Atherosclerosis

Received Date: 18 March 2013

Revised Date: 2 March 2014

Accepted Date: 26 March 2014

Please cite this article as: Schrottmaier W, Oskolkova OV, Schabbauer G, Afonyushkin T, MicroRNAmiR-320a modulates induction of HO-1, GCLM and OKL38 by oxidized phospholipids in endothelialcells., Atherosclerosis (2014), doi: 10.1016/j.atherosclerosis.2014.03.026.

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ACCEPTED MANUSCRIPTMicroRNA miR-320a modulates induction of HO-1, GCLM and OKL38 by oxidized

phospholipids in endothelial cells.

Waltraud Schrottmaier2, Olga V. Oskolkova1, Gernot Schabbauer2* and Taras Afonyushkin1*.

1Department of Vascular Biology and Thrombosis Research, Centre for Physiology and

Pharmacology, Medical University of Vienna, Vienna, Austria.

2Institute for Physiology, Centre for Physiology and Pharmacology, Medical University of

Vienna, Vienna, Austria.

*Correspondence to:

Taras Afonyushkin, PhD,

Dept. of Vascular Biology and Thrombosis Research,

Centre for Physiology and Pharmacology

Medical University of Vienna,

Schwarzspanierstrasse 17,

A-1090 Vienna, Austria

Tel: +43-1-40160-31-116

Fax: +43-1-40160-93-1129

e-mail: taras.afonyushkin@meduniwien.ac.at

and

Gernot Schabbauer, PhD

Inst. for Physiology,

Centre for Physiology and Pharmacology

Medical University of Vienna,

Schwarzspanierstrasse 17,

A-1090 Vienna, Austria

Tel: +43-1-40160-31-427

Fax: +43-1-40160-93-1101

e-mail: gernot.schabbauer@meduniwien.ac.at

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Objective: Oxidized phospholipids (OxPLs), which are highly abundant in atherosclerotic

lesions, are known to induce electrophilic stress response (ESR). ESR induces

cytoprotective genes via the NF-E2-related factor 2 (NRF2) transcription factor. In order to

get further insight into the mechanisms of ESR, we studied the role of microRNA (miR)-320a

in induction of NRF2-dependent genes by OxPLs.

Methods: Microarray profiling and qRT-PCR methods were used for measurements of

mRNA and miRNA levels. miR-320a levels were changed by transfection with synthetic

oligonucleotides. Protein analysis was performed by Western blotting. The functional activity

of NRF2 was measured by DNA-binding ELISA.

Results: Oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) induced miR-320a

in endothelial cells. Induction of HO-1, OKL38 and GCLM mRNAs by OxPAPC and

sulforaphane was attenuated upon knockdown of miR-320a. In contrast, transfection of ECs

with miR-320a mimic oligonucleotide potentiated the effects of OxPAPC and sulforaphane on

induction of HO-1, OKL38 and GCLM mRNAs. OxPAPC-induced p38 activation, levels of

NRF2 protein and its ability to bind to consensus NRF2 DNA binding site were elevated in

ECs tranfected with miR-320a mimic. miR-320a positively regulated induction of VEGF

mRNA by OxPAPC. Levels of miR-320a and HO-1 and OKL38 mRNAs were elevated in

aortas of ApoE knockout mice fed with high fat diet. Manipulation of miR-320a level in ECs

did not affect ability of OxPAPC to induce IL-8, COX-2 and MCP-1.

Conclusion: miR-320a plays important role in induction of expression of HO-1, GCLM and

OKL38 upon ESR induced either by OxPAPC or sulforafane. These observations propose a

general role of miR-320a in control of ESR induced by different electrophilic agents.

Keywords

Oxidized phospholipids, electrophilic stress response, microRNA-320a, HO-1, GCLM,

OKL38, NRF2.

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Abbreviations

ARE, antioxidant response element; ECs, endothelial cells; ESR, electrophilic stress

response; GCLM, glutathioncysteine ligase modifying subunit; HO-1, heme oxygenase-1;

miRNA, microRNA; NRF2, NF-E2-related factor; OKL38, oxidative stress induced growth

inhibitor 1 ; OxPAPC, oxidized palmitoyl-arachidonoyl-phosphatidylcholine; OxPLs, oxidized

phospholipids.

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Products of phospholipids oxidation (OxPLs) are increasingly recognized to play a significant

role in development and progression of atherosclerotic disease [1]. OxPLs are present at

high concentrations in human atheroma [2]. It is known that in addition to their deleterious

effects like induction of monocytic inflammation and coagulation, OxPLs are also inducing

vasculoprotective pathways promoting compensatory reactions of endothelium to stress [1].

OxPLs have been shown to induce Phase II and antioxidant genes via electrophilic

stress response (ESR) mechanism [3]. Expression of ESR genes heme-oxygenase 1(HO-1),

glutathioncysteine ligase modifying subunit (GCLM) and oxidative stress induced growth

inhibitor 1 (OKL38) are induced at the transcriptional level upon binding of NF-E2-related

factor (NRF2) to antioxidant response element (ARE) sequence within their promoter regions

[3-5, 5]. HO-1 plays a major role in protection of endothelium against oxidative stress [6].

HO-1 deficiency or pharmacological inhibition promotes atherosclerotic lesions development

and plaque instability [6].GCLM is the rate-limiting protein in synthesis of major cellular

antioxidant glutathione. It was shown that GCLM deficiency accelerates atherosclerotic

plaque development in aortas of experimental animals [7]. OKL38 was proposed to protect

endothelium by downregulation of cyclins and through the metabolism of epoxide [5, 8].

Altogether, these data point at the importance of HO-1, GCLM and OKL38 as

atheroprotective genes.

Electrophilic compounds induce dissociation of NRF2 from KEAP1 protein, which

targets NRF2 to ubiqitination and proteosomal degradation under non-stress conditions [9].

In addition electrophilic compounds promote posttranslational modifications of NRF2

changing its stability, nuclear translocation and transcriptional activity [9]. In summary these

events lead to the activation of ARE genes expression. Activation of NRF2 by electrophilic

compounds, which is followed by induction of cytoprotective/antioxidant genes, attenuates

oxidative stress and induces atheroprotective and antiinflammatory effects [1]. However,

NRF2-ARE activation also plays a negative role by promoting advanced tumor progression

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provide new therapeutic approaches in the treatment of human diseases.

Small regulatory RNAs – microRNAs (miRNAs) regulate gene expression at the levels

of translation and/or mRNA stability. Recently, miRNAs were demonstrated to be involved in

modulation of NRF2-ARE axis: miR-200a targets KEAP1 mRNA, miR-155 downregulates

ARE-binding transcriptional repressor BACH1 and miR-28 suppress translation of NRF2

mRNA [11-13].

In the current work we performed microarray based miRNAs profiling in human

umbilical vein endothelial cells (HUVEC) stimulated with oxidized palmitoyl-arachidonoyl-

phosphatidylcholine (OxPAPC) and observed significant upregulation of miRNA-320a. miR-

320a was previously reported to be induced by oxidative stress [14]. Similarly to oxidative

stress inducers and electrophiles OxPAPC is also known to induce expression of

cytoprotective genes like HO-1 and GCLM [3]. Here we demonstrate that miR-320a positively

modulated the basal and OxPAPC-stimulated expression of HO-1, OKL38 and GCLM.

Similarly, induction of HO-1, OKL38 and GCLM by classical ESR agent sulforaphane is also

dependent on the miR-320a level. Furthermore, miR-320a positively affected NRF2 protein

levels via activation of the MAP kinase p38 [15] and its ability to bind to consensus ARE in

OxPAPC-stimulated HUVEC. Moreover, we could demonstrate that miR-320a might be

involved in the regulation of OxPAPC-induced expression of VEGF mRNA via the NRF2-

ATF4 axis. Collectively, our data strongly suggest that miR-320a plays important role in

activation of ARE genes in endothelial cells by OxPLs.

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Materials, cell culture and lipids preparation.

Sulforaphane and polyethylenimin (PEI) were obtained from Sigma-Aldrich. Human umbilical

vein EC (HUVEC) (Lonza) were grown at 37°C in 5% CO 2 in medium M199 containing 20%

fetal calf serum (FCS), 1 U/ml heparin, EC growth supplement (Promocell), 2 mmol/l

glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and used up to passage 5. 1-

palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) (Avanti) was oxidized by

exposure to air. Formation of OxPAPC was controlled by electrospray ionization-mass

spectrometry and thin-layer chromatography [16]. Prior to cells stimulation, the lipids were

resuspended in medium M199 containing 2% FCS.

Transfection with miRNA mimic and inhibitor

ECs were transfected with 50 nM of either miRNA mimic, miRNA inhibitor or control

oligonucleotide (all from Qiagen) in serum- and antibiotic-free M199 medium for 4 hours

using the PEI reagent, then the transfected cells were allowed to rest for at least 20h [17]. All

stimulations were performed 24 h after transfection.

Microarray profiling of miRNAs, cDNA synthesis and qRT-PCR analysis of RNA.

HUVECs were treated either with 100 µg/ml OxPAPC in medium M199 containing 2% serum

or with control medium for 4 hours. Trizol reagent (Invitrogen) was used for RNA isolation.

Microarray based profiling of microRNAs and data analysis was done by the LC Sciences.

For RNA extraction from mouse aortas sacrificed animals were perfused with PBS and

RNAlater Reagent (Ambion). Thereafter extracted aortas were homogenized and total RNA

was isolated using Trizol reagent (Invitrogen).

For mRNA analysis cDNA was synthesized from 300 ng of total RNA by using qRT-PCR

300a GeneAmp RNA-PCR kit (Applied Biosystems). Thereafter quantitative real-time PCR

was performed (95°C 3 sec, 60°C 30 sec, 30 cycles) with Fast SYBR Green Master Mix

(Applied Biosystems) according to the manufacturer`s instructions. Relative levels of miR-

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were converted into cDNA using TaqMan MicroRNA Reverse transcription Kit and RNA

specific reverse transcription primer (Applied Biosystems). TaqMan universal PCR Master

Mix II and a set of TaqMan qRT-PCR primers (all Applied Biosystems) were used for

quantification of microRNA according to the vendor manual (95°C 15 sec, 60°C 60 sec, 40

cycles). Sequences of primers are available upon request. All reactions were performed on

the StepOnePlus Real-Time PCR Cycler (Applied Biosystems).

The fold increase or decrease in individual mRNAs or miR-320a was calculated using the

standard ∆CT method. The expression of each mRNA was mormalized to β2-microglobulin

mRNA, expression of miR-320a was normalized to the U6 snRNA.

Western blotting and NRF-ARE binding assay

NE-Per Kit (Pierce) was used for preparation of nuclear and cytoplasmic protein extracts

from OxPAPC-stimulated and untreated HUVECs. Protein samples were denatured in

Laemmli buffer and separated in SDS- polyacrylamide gels with following electroblotting to

PVDF membrane (Millipore). Protein blots were probed with NRF2, KEAP1, Lamin A and

β−Tubulin antibodies (all from Santa-Cruz) and p38, phospho-p38, PTEN (all from Cell

Signaling Tech.) and ß-Actin (from Sigma). Horseradish peroxidase conjugated IgG (GE

Healtcare) and SuperSignal West Femto Substrate (Pierce) were used for detection of bound

primary antibodies. Fluorescence was detected by using FluorChem HD2 imager (Alpha

Innotech). NRF2-ARE interactions were analyzed by using ELISA - based TransAM NRF2

Kit (Active Motif). Nuclear extracts were incubated with consensus ARE immobilized on a 96-

well plate. ARE bound NRF2 was detected by NRF2 antibody and secondary horseradish

peroxidase conjugated antibody. The signal was detected spectrophotometrically at 450 nM.

Mice

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chow diet (6.5% fat) or a proatherogenic diet (15% fat, 1.25% cholesterol) (Sniff) for 8 weeks,

six animals per group. Thereafter total RNA was prepared from homogenized aortas by using

Trizol reagent (Invitrogen) and analyzed as described above. Experiments were approved by

the Medical University of Vienna animal experimentation committee and the Austrian Ministry

of Science.

Statistical analysis

GraphPad Prism Software was used for statistical analyses. Two-tailed Student's t-test was

used for analysis of data obtained in in vitro experiments and P value less than 0.05 was

considered significant. Data are represented as means ± standard deviations (n=4). Data

obtained in in vivo experiments were analyzed by ANOVA and Bonferroni Post Test. P value

less than 0.05 was considered significant. Data are represented as means (n=6).

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3.1. OxPAPC induces miR-320a in HUVEC.

Phospholipid oxidation products are increasingly recognized for their role in

development and progression of atherosclerosis [18]. Small regulatory RNAs – microRNAs –

play a significant role in the pathogenesis of vascular diseases [19]. However, knowledge

about the role of microRNAs as mediators of OxPLs effects is very limited. In addition to the

previously published miR-663, which is important for induction of ATF4 and VEGF [20], we

observed upregulation of miR-320a in OxPAPC-treated HUVEC using microarray profiling

(Fig. 1A). miR-320a is known to be induced by oxidative stress [14]. Indeed we found

upregulation of miR-320a upon OxPAPC stimulation by qRT-PCR analysis (Fig. 1B),

confirming the array data.

3.2. miR-320a modulates induction of HO-1, OKL38 and GCLM mRNAs in OxPAPC-

stimulated HUVEC.

Similarly to oxidative stress inducers OxPLs induce expression of genes containing

antioxidant response element (ARE) via activation of NRF2 transcription factor [3]. NRF2-

dependent genes HO-1 and OSGIN/OKL38 were proposed to play a key role in

cytoprotective, pro- and anti-inflammatory responses induced by OxPAPC [5, 21]. In order to

test whether miR-320a is involved in the regulation of ARE-genes, HUVEC were transfected

with oligonucleotides mimicking (m320a) or inhibiting (i320a) miR-320a, or with control (Co)

oligonucleotide.

Transfection with m320a and i320a led to significant decrease and increase of miR-

320a levels, respectively (Supplementary Fig. 1A and 1B). We observed that i320a

attenuated and m320a potentiated significantly the induction of HO-1, OKL38 and GCLM

mRNA levels in OxPAPC-treated HUVEC (Fig1C, D and E). In contrast, neither i320a nor

m320a had any effect on induction of IL-8, MCP-1 and COX-2 mRNAs (Supplemetary Fig.

2A-C), suggesting that miR-320a specifically modulates ARE-genes expression. Moreover,

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unstimulated HUVEC (Fig. 1C-E).

3.3. miR-320a positively regulates NRF2 levels and NRF2-DNA binding.

Induction of HO-1, OKL38 and GCLM ARE genes upon ESR induced by OxPAPC is known

to be under the control of NRF2 transcription factor [3, 4]. We performed analysis of binding

of nuclear extracts prepared from OxPAPC-stimulated HUVEC transfected with i320a or

m320a to the oligonucleotide corresponding to the consensus ARE site and observed

positive and negative effects of m320a and i320a on NRF2-ARE binding, respectively (Fig.

2A). These data suggest that miR-320a modulate induction of HO-1, OKL38 and GCLM via

affecting NRF2 transcription factor. In order to check whether miR-320a may affect induction

of NRF2 by OxPAPC, we analysed levels of NRF2 in cytoplasmic and nuclear extracts from

HUVEC transfected with i320a, m320a or control oligonucleotides by Western blotting.

Elevation of NRF2 protein in nuclear extracts upon OxPAPC stimulation was

increased by m320a and attenuated by i320a (Fig. 2B). m320a transfection also led to the

elevated accumulation of NRF2 in the cytoplasm (Fig. 2C). KEAP1 protein is known to

negatively regulate NRF2 levels by promoting its ubiqitination [22]. We observed no effect of

i320a and m320a on the KEAP1 protein (Fig. 2C) and on the NRF2 and KEAP mRNAs levels

(Supplementary Fig. 3A and B). The NRF2/KEAP interaction is also regulated either by

activation of p38 or the PI3K/PTEN signalling pathway [15]. Indeed we found an increase in

p38 phosphorylation upon transfection with m320a, without affecting PTEN levels, which

indicates an effect of the MAPK signalling activation on the release of NRF2 from KEAP1

(Fig. 2D).

These data support the notion that both NRF2 and KEAP1 are not direct targets of

miR-320a and allow hypothesizing that miR-320a modulate NRF2 induction by affecting

genes involved in its posttranslational modifications.

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ApoE knockout mice fed with atherogenic diet.

OxPLs are known to accumulate in vessels of patients with atherosclerotic desease [2] and

dyslipidemic ApoE knockout mice fed with atherogenic diet [23]. In order to check whether

the levels of miR-320a are elevated in vivo we analysed RNAs isolated from apoE and wild-

type mice fed with the chow or high-fat diet by qRT-PCR. We observed statistically significant

elevation of miR-320a in aortas of ApoE knockout mice fed with high fat diet versus wild type

mice fed with the chow diet (Fig. 3A). Admittedly, analysis revealed only a trend for

differential upregulation of miR-320a, HO-1, OKL38 and GCLM, when we compared the data

sets within the groups of wild-type mice as well as the ApoE deficient dyslipidemic mice.

HO-1 is known to be induced by OxPLs in endothelial cells in vitro [24] and elevated in aortas

of experimental animals fed high fat diet [25, 26]. In good agreement with published data we

found significant increase of HO-1 mRNAs in aortas of ApoE knockout animals fed

atherogenic diet Fig. 3B and C. mi320a levels correlated with elevated levels of HO-1,

OKL38 and GCLM mRNAs (Fig. 3A- D).

3.5. Induction of HO-1, GCLM and OKL38 mRNAs by sulforaphane depends on miR-320a.

In order to answer the question whether the effects of miR-320a on induction of ARE genes

are unique for OxPLs, we used the classical inducer of ESR sulforaphane known to induce

NRF2 transcriptional activity [27]. We observed that similarly to OxPAPC induction of HO-1,

OKL38 and GCLM mRNAs by sulforaphane was attenuated by miR320a inhibitor and

potentiated by miR-320 mimic (Fig. 4A-C). In addition we tested the possibility that OxPAPC

or sulforaphane down-modulatory effects on TNFα-induced genes are modulated by

miR320a [18, 28]. We observed that both OxPAPC and sulforaphane pretreatment blocked

induction of VCAM and E-selectin mRNAs by TNFα (sFig. 4A and B). However both,

miR320a inhibitor as well as miR-320 mimic, did not influence the ability of sulforaphane and

OxPAPC to inhibit induction of VCAM and E-selectin mRNAs by TNFα. These data suggest

that miR-320a plays a general role in modulation of the NRF2-ARE axis but is not causally

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3.6. miR-320a positively regulates expression of ATF4, VEGF and TRB3 mRNAs.

Induction of VEGF mRNA by OxPAPC and is known to be driven by ATF4 transcription factor

[29] and induction of ATF4 mRNA by OxPAPC is under transcriptional control of NRF2 [30].

In order to answer the question whether miR-320a is involved in the regulation of the NRF2-

ATF4-VEGF axis, we analysed the effects of artificial manipulation of miR-320a level on the

induction of ATF4 and VEGF mRNAs by OxPAPC. Transfection of cells with i320a and

m320a attenuated and potentiated respectively induction of ATF4 and VEGF mRNAs (Fig.

5A and B). In good agreement with these data we observed same effect of miR-320a on

mRNA expression of another transcriptional target of ATF4, TRB3 (Fig. 5C).

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Cytoprotective ARE genes like HO-1 and GCLM are known to play important roles in

maintaining endothelial cell function under physiological and pathophysiological conditions.

These genes are known to be activated at the transcriptional level by NRF2 [31]. Key

antioxidative genes, such as HO-1 and GCLM, were demonstrated to be expressed in

atherosclerotic plaques [26]. Deficiency of HO-1 or GCLM accelerates atherosclerotic plaque

development in experimental models of atherosclerosis [6, 7]. OKL38 was proposed to play

endothelium protective role [5]. Moreover it is a key regulator of expression of pro- and anti-

inflammatory genes [21]. Therefore, approaches aimed to modulating ARE genes expression

are widely recognized as a promising therapy of cardiovascular diseases [32]. The major

finding of the present study is that microRNA-320a positively regulates expression of ARE

genes HO-1, GCLM and OKL38 in endothelial cells. Thus, miR-320a may be used as a novel

tool for modulation of vasculoprotective ARE genes expression.

In addition to their atherogenic, proinflammatory and prothrombogenic action, OxPLs

are also capable of inducing expression of HO-1, GCLM, OKL38 and NQO by activating

transcription factor NRF2 [3, 4]. Moreover, HO-1 and OKL38 were proposed to play a critical

role in activation of ECs by oxidized phospholipids [33]. In the current study we demonstrated

that OxPAPC induced miR-320a in ECs and that miR-320a positively regulated basal and

OxPAPC-induced levels of HO-1, GCLM and OKL38 mRNAs. These results underscore the

importance of miR-320a in ESR induced by OxPLs.

We did not observe any significant effect of miR-320a on the levels of IL8, COX-2, and

MCP-1 mRNAs induced by OxPAPC. Induction of IL-8 by OxPAPC is mediated by SREBP

and STAT3 [34]. Previously we have shown stabilisation of COX-2 transcript upon OxPAPC

stimulation which leads to increase of COX-2 mRNA level [29]. p38 MAPK plays critical role

in induction of MCP-1 mRNA by OxPLs [35]. These data illustrate the specificity of miR-320a

on the modulation of ARE genes.

Several microRNAs are known to affect expression of ARE genes: miR-155

downregulates translation of ARE-binding transcriptional inhibitor BACH1 [12], while miR-28

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these findings, miR-320a did not affect levels of KEAP1 mRNA/protein and NRF2 mRNA

levels. Observation that miR-320a which is known to be induced by oxidative stress [14]

positively modulated NRF2 protein level and ability of NRF2 to bind to consensus ARE upon

stimulation with OxPAPC allow to hypothesize that miR-320a induces expression of ARE

genes by inducing of NRF2 rather than inhibiting BACH1. The absence of effect of miR-320a

on NRF2 mRNA abundance excludes the possibility of transcriptional regulation of NRF2

gene expression by miR-320a. The observation that miR-320a positively regulates level of

NRF2 protein both in cytoplasm and nuclei of unstimulated cells allows us to propose that

miR-320a positively affects NRF2 protein stability rather than NRF2 nuclear translocation.

This is clearly supported by the enhanced p38 MAP kinase activation, which is linked to

NRF2/KEAP dissociation and NRF2 activation [15]. Thus, we hypothesize that miR-320a

positively regulates expression of HO-1, OKL38 and GCLM mRNAs by affecting mechanisms

of posttranslational modification of NRF2 transcription factor. NRF2 is known to be a target of

several posttranslational modifications, which may modulate its stability, activity and nuclear

localization [9, 15]. Further studies are definitely required to get more insight in mechanisms

of miR-320a-NRF2 interaction and regulation.

OxPLs are known to accumulate in vessels of patients with atherosclerotic disease and

aortas of ApoE knockout mice fed with atherogenic diet [2, 23]. Another important finding of

the current work is that miR-320a is elevated in aortas of ApoE knockout animals fed with

atherogenic diet. This observation strongly points to possible role of miR-320a in endothelial-

protective mechanisms in the course of atherosclerosis.

We found that miR-320a modulates induction of HO-1, OKL38 and GCLM mRNAs by

sulforaphane. Sulforaphane is a classical inducer of ESR and is chemically unrelated to

OxPLs. Therefore, we hypothesize that regulation of ARE genes by miR-320a is not limited

to specific electrophilic compounds such as OxPLs. We propose that miR-320a plays a more

general role in modulation of ESR.

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therefore artificial modulation of miR-320a may provide new perspectives in treatment of

atherosclerotic disease. In order to confirm this notion, long-term effects of miR-320a have to

be tested in in vivo models of atherosclerosis. Also prospective analysis of miR-320a in

plasma and vessels of patients with cardiovascular diseases and control subjects is of high

interest.

Previously we have shown that induction of VEGF expression by ATF4 transcription

factor in OxPL-stimulated ECs critically depends on NRF2 [30]. Here we demonstrate that

miR-320a positively regulates induction of ATF4 mRNA and ATF4-dependent VEGF and

TRB3 mRNAs by OxPAPC. Thus, our former and present findings allow us to hypothesize

that the miR-320a might be involved in regulation of VEGF expression and thereby might

affect OxPLs-induced angiogenesis.

Although the focus of this work is on the role of miR-320a in induction of ARE genes in

ECs, we propose that miR-320a-NRF2-ARE axis may play important role in other cell types

and disease states. NRF2-driven cytoprotective genes are important in the inhibition of

carcinogenesis [10]. However, NRF2 induction plays deleterious role in advanced tumors by

promoting chemoresistance and growth [10].

In conclusion, our work demonstrates an important role for miR-320a in the regulation

of ARE genes in the course of electrophilic stress response induced by atherogenic

phospholipids or sulforaphane in endothelial cells, thus providing further insights in

mechanisms controlling and mediating ESR.

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The work was supported by the grants from Fonds zur Förderung wissenschaftlicher

Forschung (P23016-B11 to TA and P22267-B11 to O.V.O).

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ACCEPTED MANUSCRIPTFigure legends

Figure 1. miR-320a is induced in HUVECs by OxPAPC and modulates induction of HO-

1, GCLM and OKL38 mRNAs.

HUVECs were stimulated with 100 µg/ml OxPAPC for 4 hours. A. Microarray data on

OxPAPC-regulated microRNAs. B. miR-320a expression in HUVECs stimulated with

OxPAPC was quantified by real-time qRT-PCR and normalized to U6 snRNA levels. C, D

and E. The levels of HO-1, GCLM and OKL38 mRNAs were quantified in cells transfected

with control, miR-320a inhibiting or miR-320a mimicking oligonucletides (50 nM, 24h) by real-

time qPCR and normalized to β2-microglobulin mRNA.

Figure 2. miR-320a positively regulates p38 activation, NRF2 level and NRF2-DNA

binding.

HUVECs transfected with control, miR-320a inhibiting or mimicking oligonucleotides (50 nM,

24h) were treated with 100 µg/ml OxPAPC for 2 hours or 30 minutes in case of p38

phosphorylation. A. NRF2-DNA binding activity of nuclear extracts from HUVECs stimulated

with OxPAPC. 2 mg of nuclear extracts were incubated with immobilized consensus ARE,

amount of bound NRF2 was measured using the ELISA-based TransAM NRF2 kit. B.

Western blotting analysis of effects of miR-320a on OxPAPC-induced NRF2 levels in nuclear

extracts, lamin B1 served as a loading control. C. Effect of miR-320a on the levels of NRF2,

KEAP1 and tubulin proteins in cytoplasm upon stimulation with OxPAPC was measured by

Western blotting. D. Effect of miR-320A on the activation of p38 MAP kinase by stimulation

with OxPAPC after 30 minutes was measured by Western blotting.

Figure 3. Levels of miR-320a and HO-1, OKL38 and GCLM mRNAs are elevated in

aortas of apoE knockout mice fed with high-fat (atherogenic) diet.

4 months old wild-type and apoE knockout mice were fed with control or high-fat diet for 8

weeks. Total RNA was isolated from aortas and analyzed by real-time qRT-PCR. U6 snRNA

and β2-microglobulin mRNA levels were used for normalization.

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Figure 4. Induction of HO-1, OKL38 and GCLM mRNAs by sulforaphane is regulated by

miR-320a.

A, B and C. Levels of HO-1, GCLM and OKL38 mRNAs were quantified by real-time qRT-

PCR and normalized to β2-microglobulin mRNA. HUVECs were transfected with 50 nM

control, miR-320a mimicking or miR-320a inhibitory oligonucleotides. 24 hours after

transfection cells were stimulated for 4 hours with 5 µg/ml sulforafane

Figure 5.. miR-320a positively regulates expression of ATF4, VEGF and TRB3 mRNAs.

A, B and C. Levels of ATF4, VEGF and TRB3 mRNAs were quantified by real-time qRT-PCR

and normalized to β2-microglobulin mRNA. HUVECs were transfected with 50 nM control,

miR-320a mimicking or miR-320a inhibitory oligonucleotides. 24 hours after transfection cells

were stimulated for 4 hours with 100 µg/ml OxPAPC for 4 hours.

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Supplementary Figure legends

Supplementary Figure 1. Transfection with mimicking and inhibitory

oligonucleotides changes the level of miR-320a.

A: Transfection of HUVECs with miR-320a inhibitory oligonucleotide (i320a) (50 nM, 24

hours) reduces levels of miR-320a. B: miR-320a is elevated upon transfection of

HUVECs with miR-320a mimic oligonucleotide (m320a) (50 nM, 24 hours). miR320a

was quantified by qRT-PCR and data were normalized to U6 snRNA.

Supplementary Figure 2. Overexpression or knockdown of miR-320a do not affect

induction of NRF2-independent genes IL8 and COX-2.

HUVECs were transfected with miR-320a inhibitory (i320a) or mimic oligonucleotides

(m320a) (50 nM) using PEI reagent. 24 hours later, the cells were treated with OxPAPC

(100 µg/ml, 4 h) followed by quantification of IL8 (A) and COX-2 (B) mRNAs using RT-

PCR with normalization to the ß2-microglobulin mRNA.

Supplementary Figure 3. Knockdown and overexpression of miR-320a did not

affect NRF2 and KEAP1 mRNA levels.

HUVECs were transfected with miR-320a inhibitor (i320a), mimic (m320a) or control

oligonucleotides (50 nM, 24h). Thereafter, cells were stimulated with OxPAPC (100

µg/ml, 4 h). Level of NRF2 (A) and KEAP1 (B) was analyzed by qRT-PCR and

normalized to ß2-microglobulin mRNA.

Supplementary Figure 4. Ability of OxPAPC and sulforaphane to inhibit induction

of VCAM and E-selectin by TNFαααα is not affected by miR-320a.

mRNA levels of VCAM (A), E-selectin (B) were measured by real-time qRT-PCR and

normalized to β2-microglobulin mRNA level. HUVECs were transfected with 50 nM

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control, miR-320a mimicking or miR-320a inhibitory oligonucleotides. 24 hours after

transfection cells were pretreated with OxPAPC (100 µg /ml) or sulforaphane (5 µg/ml)

for 30 minutes. Thereafter cells were stimulated with 20 ng/ml TNFα for 4 hours.

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P<0.01

P<0.01

Figure 1

A

OxPAPC+ + +_ _ _ 0

1

2

3

Co OxPAPC

B

miR

-320

a R

NA

, fol

d of

con

trol P<0.05

C

D

E

020406080

100120140160180

0

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ControlOxPAPC

+_

i320a+__ m320a_

+_+__ _

+_

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+_+__ _

+_

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+_+__ _

ControlOxPAPC

ControlOxPAPC

P<0.05

P<0.01

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P<0.05

P<0.01 P<0.001

P<0.01

P<0.01

P<0.01

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Figure 2

++_

OxPAPC_ _

+

B

NRF2

Lamin B

C++

_OxPAPC

_ _+

NRF2

KEAP1

A

β−tubulin

i320am320a+_

+ __ _

+_

+__ _

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0

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Figure 3A B

C D

0

0.5

1

1.5

2

2.5Chow High fat

wt wt ApoE ApoE0

5

10

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25Chow High fat

wt wt ApoE ApoE

0

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10

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wt wt ApoE ApoE 0

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wt wt ApoE ApoE

miR

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i320a+_

+__ m320a_

+_+__ _

Figure 4

AA

HO

-1 m

RN

A, f

old

of c

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ControlSulforaphane

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P<0.05 P<0.05

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F4

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,fold

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012345678

0

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8

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B

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Supplementary Figure 1

A

B

P<0.05

0

0.2

0.4

0.6

0.8

1

1.2

1,4

i320aComiR

-320

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B

Supplementary Figure 2

A

5101520253035404550 Control

OxPAPC

0+_

i320a+__ m320a_

+_+__ _

05

1015202530354045

CO

X-2

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ControlOxPAPC

IL8

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B

00.20.40.60.81

1.21.41.61.8 Control

OxPAPC

KE

AP

1 m

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A, f

old

of c

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+_

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+_+__ _

A

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4.5 ControlOxPAPC

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10001500200025003000

0200400600800

1000

4000

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TNFα

i320Co m320

ns

ns

+_ _ _ + + +_ _ _ + + +_ _ _ + +_ __ ++ _ _ __ ++ _ _ __ ++ __ __ ++ _ _ __ ++ _ _ __ ++ _

OxPAPCSulforaphane

A

B

TNFα

i320Co m320

ns

ns

+_ _ _ + + +_ _ _ + + +_ _ _ + +_ __ ++ _ _ __ ++ _ _ __ ++ __ __ ++ _ _ __ ++ _ _ __ ++ _

VC

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d of

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OxPAPCSulforaphane

E-s

elec

tin m

RN

A, f

old

of c

ontr

olSupplementary Figure 4

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miR-320a is induced in endothelial cells upon OxPAPC treatment. miR-320a positively regulates induction of HO-1, GCLM and OKL38 by OxPAPC and sulforaphane in endothelial cells miR-320a is critically important for induction of transcription factor NRF2 by OxPAPC.