The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor

    The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor Nakwon Choe, Jin-Sook Kwon, Yong Sook Kim, Gwang Hyeon Eom, Young Keun Ahn, Yung Hong Baik, Hyun-Young Park, Hyun Kook PII: DOI: Reference:

S0898-6568(15)00035-2 doi: 10.1016/j.cellsig.2014.12.022 CLS 8392

To appear in:

Cellular Signalling

Received date: Revised date: Accepted date:

22 September 2014 9 December 2014 26 December 2014

Please cite this article as: Nakwon Choe, Jin-Sook Kwon, Yong Sook Kim, Gwang Hyeon Eom, Young Keun Ahn, Yung Hong Baik, Hyun-Young Park, Hyun Kook, The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor, Cellular Signalling (2015), doi: 10.1016/j.cellsig.2014.12.022

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The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal

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hyperplasia by targeting stem cell factor

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Short title: miR-34c targets SCF to inhibit neointima

Yung Hong Baik5, Hyun-Young Park3, Hyun Kook1,2

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Nakwon Choe1,2, Jin-Sook Kwon3, Yong Sook Kim4, Gwang Hyeon Eom1,2, Young Keun Ahn4,

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Medical Research Center for Gene Regulation1 and Department of Pharmacology2,Chonnam

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National University Medical School, Gwangju 501-746, Republic of Korea Division of Cardiovascular and Rare Disease, Korea National Institute of Health3, Osong,

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Cheongju, Chungbuk, Korea, 363-951, Republic of Korea

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Department of Cardiology4, Chonnam National University Hospital, Gwangju 501-757, Republic

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of Korea

Department of Pharmacology5, College of Medicine, Seonam University, Namwon, 590-711, Republic of Korea

Corresponding author: Hyun Kook, MD PhD Department of Pharmacology, Medical Research Center for Gene Regulation and National Research Laboratory for Heart and Muscle Diseases Chonnam National University Medical School 5 Hak-dong, Dong-ku, Gwangju, 501-746 South Korea

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+82-62-220-4242 (office) +82-62-220-4243 (lab)

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+82-62-232-6974 (fax)

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e-mail: [email protected]

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Abstract

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The fine balance between proliferation and differentiation of vascular smooth muscle cells

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(VSMCs) is indispensable for the maintenance of healthy blood vessels, whereas an increase in proliferation participates in pathologic cardiovascular events such as atherosclerosis and

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restenosis. Here we report that microRNA-34c (miR-34c) targets stem cell factor (SCF) to inhibit

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VSMC proliferation and neointimal hyperplasia. In an animal model, miR-34c was significantly increased in the rat carotid artery after catheter injury. Transient transfection of miR-34c to either

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VSMCs or A10 cells inhibited cell survival by inducing apoptosis, which was accompanied by

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an increase in expression of p21, p27, and Bax. Transfection of miR-34c also attenuated VSMC migration. Bioinformatics showed that SCF is a target candidate of miR-34c. miR-34c

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down-regulated luciferase activity driven by a vector containing the 3’-untranslated region of SCF in a sequence-specific manner. Forced expression of SCF in A10 cells induced proliferation and migration, whereas knocking-down of SCF reduced cell survival and migration. miR-34c antagomir-induced VSMC proliferation was blocked by SCF siRNA. Delivery of miR-34c to rat carotid artery attenuated the expression of SCF and blocked neointimal hyperplasia. These results suggest that miR-34c is a new modulator of VSMC proliferation and that it inhibits neointima formation by regulating SCF.

Keywords: miR-34c, stem cell factor, vascular smooth muscle cells, neointimal hyperplasia, atherosclerosis

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1. Introduction

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The characteristics of atherosclerosis include the thickening of intima resulting from the

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accumulation of vascular smooth muscle cells (VSMCs) and inflammatory cells and the

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deposition of extracellular matrix. The complex interaction between VSMCs and endothelial cells or various types of lymphocytes results in VSMC proliferation and migration and

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extracellular matrix synthesis. The balance between proliferation and differentiation of VSMCs

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is critical for maintaining healthy blood vessels. However, the detailed mechanisms of these phenotypic alterations and the relationship between phenotypic switching and atherosclerosis

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remain to be investigated.

MicroRNAs (miRs) are single-stranded noncoding RNAs that regulates gene expression at the

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post-transcriptional level by binding to the 3’-untranslated region (3’-UTR) of the mRNAs of their target genes. Because a perfect match between the miR and the target mRNA is not required, individual miRs can target multiple mRNAs, and a single mRNA can be regulated by multiple miRs. Given that miRs often modestly repress the expression of many mRNAs, up to90% of human genes and related cellular functions are under their control[1].

miRs mediate various cellular functions including proliferation[2], apoptosis[3],differentiation[4], and migration[5]. Previous studies have demonstrated that miR-21[6], miR-145[7], miR-221 and miR-222[8] modulate VSMCs in neointimal hyperplasia and lesion formation. While miR-34c is involved in diverse cellular process in the cardiovascular system[9, 10], the detailed mechanism of the function of miR-34c in VSMC proliferation and atherosclerosis is not yet fully understood.

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In the present study, we aimed to identify the role of miR-34c in neointimal hyperplasia. The

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effect of miR-34c was investigated in vitro and in vivo along with stem cell factor (SCF), also

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known as Kit-ligand (KITLG), which is a putative target of the miR. SCF, a ligand for the

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proto-oncogene c-kit, has been reported to be expressed in vascular endothelial cells, smooth muscle cells[11], and wire-injured mouse femoral artery[12]. Along with SCF, expression of its

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receptor c-Kit has been found to be significantly increased within the injured rat carotid

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artery[13].

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We hypothesized that miR-34c could interfere with neointimal VSMC growth by inhibiting SCF.

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The effects of miR-34c and Scf were investigated in VSMC. Proliferation, apoptosis, and

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migration were observed after either miR-34c or SCF had been up or down regulated in VSMC. Moreover, local delivery of miR-34c mimic and rat carotid artery balloon injury model were employed in order to down-regulated SCF in injured blood vessel, and subsequently the expression level of SCF and the formation of neointima were monitored.

2. Materials and Methods

All experimental procedures were approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee.

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2.1. Microarray analysis of microRNA expressionin rat carotid artery after balloon injury

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Male Sprague–Dawley rats weighing approximately 250 g were anesthetized with an

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intraperitoneal injection of ketamine (50 mg/kg) and xylazine (6.7 mg/kg). Balloon injury was

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induced in the carotid arteries of the rats as described previously[14], and arterial samples were obtained 1, 7, and 14 days later. Total RNA was extracted from the injured arterial sections with

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Trizol (Molecular Research Center Inc.; RT 111) reagent, and the miR expression profile of the

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sample was analyzed by GenomicTree Inc. by use of the Agilent miRNA Microarray System (Agilent Technologies; ID: G4473B). The expression of individual miRs was further confirmed

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2.2. Cell culture

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by real-time quantitative RT-PCR.

The smooth muscle cell line A10 (CRL-1476), derived from the thoracic aorta of rat embryo, was obtained from ATCC (Manassas, VA, USA) and was maintained in Dulbecco’s modified Eagle’s medium (Thermo Scientific; SH30243.01) supplemented with 10% fetal bovine serum (Thermo Scientific; SH30532.03). Primary cultures of VSMCs were established from rat aortas as described previously[14].

2.3. Quantitative real-time RT-PCR

Quantitative analysis of miRNA was performed by using Quantitect Real Time PCR Mix (Qiagen; catalog no. 204143) and a Rotor-Gene Q real-time PCR cycler (Qiagen). The

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sequence-specific primers for reverse transcription and quantitative real time RT-PCR of miR-34cand 18S rRNA were purchased from Applied Biosystems. The concentration of

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miR-34was normalized against 18S rRNA, and GAPDH was used as a housekeeping gene for

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calculating the expression level of the other genes.

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2.4. Western blot analysis

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Protein samples were analyzed following protocol described previously[9] using antibodies against SCF (Abcam, ab64677), ERK (Santa Cruz Biotechnology, sc-7383), AKT (Cell Signaling,

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#9272), p21 (Abcam, ab7960), p27 (Abcam, ab7961), KLF4, Bcl2 (Santa Cruz Biotechnology,

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sc-492), Bax (Santa Cruz Biotechnology, sc-526), and GAPDH (Sigma, G9545) at a 1:1000

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dilution. The image was acquired using Luminescent Image Analyzer LAS-3000 with Image Reader LAS3000 software (Fujifilm Medical Systems. Stamford, USA). Quantification of Western blot analysis was performed after retrieving the density of the bands using Scion Image software (Scion Corporation. Frederick, USA) after more than 3 independent sets of experiments.

2.5. miR mimic, antagomir, and siRNA

miR-34c mimic and antagomir, siRNA for SCF, and control si-RNA (scramble) were purchased from Bioneer Corp (Daejeon, Korea).

2.6. Cloning

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The coding sequence of rat SCF was cloned onto pcDNA6/myc-His vector for overexpression of

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SCF in mammalian cells. A DNA fragment corresponding to the 3’-UTR of SCF mRNA

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containing the putative biding site for miR-34c was cloned into pSYC-31 luciferase vector for

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luciferase assay.

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2.7. Cell proliferation in vitro

In vitro proliferation of A10 and rat VSMCs was determined by direct counting,

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and

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5-bromo-2'-deoxyuridine (BrdU) assay by using BrdU(Sigma) and anti-BrdU antibody (Santa

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Cruz Biotechnology; catalog no. IIB5).

2.8. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

Apoptosis of the A10 cells transfected with either miR-34cmimic or SCF-pcDNA6/myc-His was compared with that of cells transfected with scramble and empty vector, respectively, by using the DeadEnd™ Fluorometric TUNEL System (Promega catalog no. G3250). Parent cells serum-starved for 2 days were used as a positive control. Quantification of BrdU uptake and TUNEL positive cells were performed as following. The fluorescent signal was acquired using Nikon Eclipse microscope with Nikon DSR1 camera (Nikon Instruments Korea Co., Ltd. Seoul, Korea). Six to eight different fields per each sample were randomly chosen, the positive cells were counted in each field, the numbers were averaged, and then the averaged value was counted

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as one case. At least three independent sets of immunocytochemistry were performed for the

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quantification.

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2.9. Migration assay

The effect of miR-34c and SCF on the migration of A10 cells was observed by using a

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Transwell® (Corning Inc. Tewksbury, USA) and wound healing assay. The cell cycles of

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VSMCs were synchronized by serum-starvation for 24 hours and submitting to full confluency[15], and then serum and oxidized low-density lipoprotein (ox-LDL) (KALEN

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Biomedical; catalog no. 770202) were added to the media to induce migration. Wound healing

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assay was performed following published method[16]. In brief, cell monolayers, in the wells of

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12-well plate, were scratched with a sterile pipette tip across the diameter to create ‘wound’. The images of wound area closure acquired using Nikon Eclipse microscope with Nikon DSR1 camera were analyzed by Nikon NIS-Elements AR software (Nikon Instruments Korea Co., Ltd. Seoul, Korea). The percentage of migration rate was calculated as follows:

Migration (%) = (Area0  Areat)/Area0  100%. (Area0 = the area of day 0, Areat = the area of the indicated day)

Transwell® migration assay was used following manufacturer’s protocol. The cells migrating through Transwell® membrane were fixed with 3.7% paraformaldehyde. The images of the migrating cells stained with crystal violet were obtained using Nikon Eclipse microscope with

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Nikon DSR1 camera. The number of migrating cell was counted with ImageJ software (National

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Institute of Health. Bethesda, USA).

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2.10. Target scan

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Putative target mRNAs of miR-34cwere screened on http://www.targetscan.org/,

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http://www.microrna.org, and http://mirdb.org/.

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2.11. Luciferase assay

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A DNA fragment corresponding to the 3’-UTR of SCF mRNA was cloned into pSYC-31

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luciferase vector. The luciferase vector was co-transfected with miR-34c mimic into A10 cells, and luciferase activity was measured by using the Luciferase Assay System (Promega; E1500).

2.12. Immunohistochemistry

Arterial sections from the rat balloon injury model were analyzed by hematoxylin and eosin (H & E) staining and fluorescent immunohistochemistry with anti-SCF antibody (Abcam; ab64677) at a 1:1000 dilution. For quantification of SCF amounts, SCF immunopositivity was measured as following. The image acquired using Nikon Eclipse microscope with Nikon DSR1 camera was analyzed by Nikon NIS-Elements AR software (Nikon Instruments Korea Co., Ltd. Seoul, Korea). Eight different fields were randomly chosen each from intimal and luminal area, the fluorescent intensity from each field was measured, the numbers were averaged, the fluorescent

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intensity from luminal area was subtracted from that of intimal area, and then the subtracted value was counted as one case. Four sets of immunohistochemistry were performed for the

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quantification.

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2.13. Local delivery of miR-34c into injured rat carotid artery

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The miR-34c mimic was locally delivered into rat carotid artery immediately after balloon injury

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by use of LipofectamineRNAiMAX reagent (Invitrogen; 13778-075) and F-127 pluronic gel (Sigma P2443) as described previously[14]. Briefly, miRs and the transfection reagent were

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diluted in DMEM and then combined before being injected into injured artery. After 30 minutes,

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the transfection mixture preloaded into pluronic gel was applied around the wounded segment.

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The parameters for the severity of atherosclerosis was determined by measuring luminal area, medial area (the area inside external elastic lamina-the area inside internal elastic lamina), neointimal area (the area inside internal elastic lamina-luminal area), neointimal area /medial area ratio, and percentage stenosis

2.14. Statistics

Data are presented as mean± S.E.M. Statistical significance was determined by Student’s t-tests or one-way ANOVA, followed by the Tukey’s honestly significant difference multiple comparison post hoc test. PASW Statistics 19 (SPSS, an IBM Company, Chicago, IL) was used for statistical analysis.

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3. Results

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3.1. Expression of miR-34cis increased in atherosclerosis

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Expression levels of miRs in rat carotid artery were determined 14 days after balloon injury by use of the Agilent Microarray. Forty-four of the 350 miRNAs tested were significantly increased

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in injured vessels compared to sham-operated vessels [data deposited on Gene Expression

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Omnibus (GEO) http://www.ncbi.nlm.nih.gov/geo/, GEO accession: ncbi-p:%3ca href=GSE45433]. Quantitative real-time RT-PCR results further confirmed the microarray

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analysis data; among the dysregulated miRs, the contents of miR-34c-5p (Fig. 1A) were

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increased approximately 4.5-fold in injured artery. We next obtained arterial samples 1, 7, and 14

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days after balloon injury and performed quantitative real-time RT-PCR analysis to observe the time course of the changes in miR-34c expression. The expression of miR-34c began to increase at 7 days after vascular injury (Fig. 1B).

3.2. miR-34cdecreases the viability and mobility of VSMCs

To examine the functional role of miR-34c in atherosclerosis, we first checked whether miR-34c affects the survival of VSMCs. Indeed, the fine balance between VSMC proliferation and differentiation is one of the critical events in the development of neointimal hyperplasia as well as in restenosis[17]. The MTT assay showed that transient transfection of miR-34c to A10 cells, a rat VSMC line[18], significantly reduced cell survival even on the first day of transfection. The survival of A10 cells gradually decreased as the treatment interval increased (Fig. 2A). Transient

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transfection of miR-34c also reduced the cell survival of VSMCs as determined by MTT assay (Fig. 2B) and direct cell counting (Fig. 2C and 2D). Transfection of scramble miR did not affect

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the cellular survival in A10 (Fig. S1A) and in VSMC cells (Fig. S1B), as determined with direct

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cell count.

BrdU uptake is an alternative parameter for determining cell proliferation[19]. miR-34c mimic

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significantly decreased BrdU uptake (Fig. 2E, middle panel in upper row). In contrast, however,

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miR-34c antagomir increased BrdU uptake (right panel). These opposite effects of mimic and antagomir on BrdU-positive cells were further quantified in Supplementary Figure 2A and 2B,

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respectively. The TUNEL assay, an indicator of apoptotic cell death[20], showed that miR-34c

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mimic increased the number of TUNEL-positive VSMCs, whereas few positive cells were seen

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when the cells were treated with the antagomir (Fig. 2E lower panels). Quantification results of mimic (Fig. S2C) and antagomir (Fig. S2D) were obtained.

We performed Western blot analysis to observe the changes in gene expression with transfection of miR-34c mimic. The expression of Bcl2, an anti-apoptotic gene[21], was decreased, whereas the expression of pro-apoptotic Bax [22] was increased. Other cell cycle regulators such as KLF4 [23], p27 [24], and p21 [25], which are known to negatively regulate cell proliferation, were upregulated by transient transfection of miR-34c in VSMCs (Fig. 2F). The effects of miR-34c mimic on Bax/Bcl2 ratio, Klf4, p27, and p21 were quantified and shown in Supplementary Figure 3.

VSMC migration is an important component of atherogenesis and neointimal formation [26].

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Thus, we investigated whether miR-34c affected VSMC migration. Transient transfection of miR-34c mimic attenuated wound closure, which represents as lowing of cell motility. In contrast,

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however, miR-34c antagomir potentiated cellular movement (Fig. 3A). The effects of miR-34c

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mimic and antagomir on cellular migration was quantified by measurement of cell-free area (Fig.

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3B), which further confirmed the negative co-relationship of miR-34c on cellular migration. A Transwell migration assay, an alternative assay for studying cell movement, also revealed that

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antagomir were further quantified (Fig. 3D).

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miR-34c negativelyregulatedA10 cell movements (Fig. 3C). The effect of miR mimic and

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3.3. miR-34c targets SCF

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We screened target candidates of miR-34cin silico to identify genes related to cell survival and proliferation. SCF, which is also known as KITLG (c-Kit ligand), was of particular interest because it was previously reported to attenuate VSMC apoptosis and to increase intimal hyperplasia in a mouse femoral artery wire-injury model [12, 27]. The alignment of miR-34c and the 3’-UTR of SCF is demonstrated in Fig. 4A. Target prediction of miR-34c was further confirmed by quantitative real-time RT-PCR for SCF. Transient transfection of miR-34c to A10 cells significantly attenuated the transcript level of SCF (Fig. 4B). Next, we generated apSYC-31-SCF 3’-UTR construct driving the luciferase gene and transfected miR-34c mimic with the UTR-plasmid to observe whether normalized luciferase activity was decreased. The miR-34c attenuated luciferase activity in a dose-dependent fashion (Fig. 4C).

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3.4. SCF induces VSMC proliferation and migration

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Next, we investigated whether SCF itself is sufficient to induce VSMC proliferation. First, we

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checked the amount of SCF after overexpression (Fig. S4A and S4C) or knocking-down (Fig.

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S4B and S4D) by measuring the protein amount. Transient transfection of a mammalian expression vector driving SCF significantly increased cell survival (Fig. 5A), whereas

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knocking-down of SCF by use of small interfering RNA technology attenuated cell survival (Fig.

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5B) in A10 cells. We further checked whether SCF could affect cellular mobilization. Both the wound healing scratch assay (Fig. 5C) and Transwell migration assay (Fig. 5D) showed that

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reduction of SCF impaired cellular movement. The quantification of migration assay was shown

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in Fig. S5.

SCF has been reported to attenuate apoptosis in human aortic smooth muscle cells treated with H2O2 and serum deprivation in an AKT-dependent manner [27]. Thus, we first checked whether SCF could induce phosphorylation of AKT, which represents activation of the AKT signal, in our experimental models. Indeed, transient transfection of pcDNA6-SCF-myc induced phosphorylation of AKT, whereas total AKT protein amounts were not altered (Fig. 5E). Interestingly, phosphorylated ERK, another downstream signaling molecule of SCF, was also increased. In addition, the protein amount of Bcl2 was increased by SCF (Fig. 5E). In contrast, when SCF was reduced by SCF siRNA, the phosphorylations of AKT and ERK and the protein amount of Bcl2 were reduced. These Western blot results were further quantified as shown in Fig. S6. These results suggest that diverse signal cascades are simultaneously involved in the SCF-induced VSMC survival in our model [28].

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3.5. Effect of miR-34c is dependent on SCF

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We next questioned whether the effect of miR-34c on cell survival was mediated by SCF. When

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miR-34c antagomir was transfected to A10 cells, as expected, cell survival was increased in 2 days. However, the proliferation was completely blocked by simultaneous transfection with SCF

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siRNA (Fig. 6A). Likewise, the miR-34c-induced increase in cellular mobility was completely

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abolished by SCF siRNA (Fig. 6B). These results suggest that, at least in part, SCF participates

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in the effects of miR-34c on cell survival and mobilization.

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3.6. Expression of SCF in atherosclerosis and blockade of neointimal hyperplasia by miR-34c

Next we checked the changes in the expression of SCF during the atherogenic process. Immunohistochemical analysis against SCF showed that the expression of SCF was increased 7 days after injury (Fig. 7A). Interestingly, however, SCF expression was lowered 7 days later, which were confirmed by measuring the fluorescence intensity of SCF after immunohistochemical analysis (Fig. 7B). We further confirmed the SCF mRNA level by quantitative real-time RT-PCR analysis. Compared with the contralateral carotid artery, SCF expression was down-regulated 1 day after injury. At 7 days, however, the mRNA level was significantly increased, which was then normalized at 14 days after injury (Fig. 7C). Compared with the expression patterns of miR-34c (Fig. 1B), the reduction of SCF at 14 days is likely to be mediated by miR-34c.

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Next, we checked whether miR-34c gene delivery resulted in a reduction of neointimal hyperplasia in an animal model. Compared with scramble-treated artery, miR-34c significantly

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reduced the neointimal hyperplasia, as shown in Fig. 8A. Quantification results showed that

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parameters for the severity of atherosclerosis, such as luminal area (Fig. 8B), neointimal area

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(Fig. 8C), neointima to media ratio (Fig. 8D), and percentage stenosis (Fig. 8E), were improved by miR-34c gene delivery. The expression of SCF was also investigated by fluorescent

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immunohistochemistry analysis. Compared with scramble-treated artery, miR-34c delivery

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resulted in a marked reduction of SCF expression (Fig. 8F).

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4. Discussion

In this article we report that the expression of miR-34c is increased in rat carotid artery after

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balloon injury and that SCF is down-regulated both in vitro and in vivo by miR-34c. Our results

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showed that miR-34c had negative effects on VSMC proliferation, survival, and migration,

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whereas its antagomir had opposite effects. Conversely, SCF had a positive effect on VSMC survival and migration, whereas inhibiting its expression with siRNA reversed the effect.

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Futhermore, knocking-down of SCF blunted the effect of the miR-34c antagomir. On the basis of these observations, we tested the hypothesis that miR-34c could interfere with neointimal VSMC

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growth by inhibiting SCF; local delivery of miR-34c mimic to rat carotid artery after vascular

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injury successfully down-regulated SCF and significantly reduced the formation of neointima.

SCF was identified as a ligand for the proto-oncogene c-Kit[28] and is known to play important roles in hematopoiesis[29], localization of melanocytes[30], and spermatogenesis[31]. It has also been reported that SCF is expressed in vascular endothelial cells and smooth muscle cells[11]. Human VSMCs express membrane-bound SCF, which is cleaved to its soluble form by metalloproteinase-9 (MMP-9), an MMP released at the time of arterial injury. Furthermore, expression of its receptor c-kit is significantly increased within the neointima in a rat carotid balloon injury model[13].Wang et al. reported that SCF is upregulated in VSMCs after vascular injury and that mutations in SCF and c-Kit attenuate neointimal hyperplasia by interrupting the SCF/c-Kit interaction required for recruitment and transformation of bone marrow progenitor cells. It has also been suggested that SCF/c-Kit attenuates vascular smooth muscle apoptosis

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through the Akt-Bcl2 pathway, which results in neointimal hyperplasia [12, 27].

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c-Kit, the receptor of SCF, is known to be linked to multiple downstream signaling pathways. In

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addition to signaling pathways involving phosphatidylinositol 3 (PI3) kinase, including Akt [27],

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SCF/c-Kit also activates mitogen-activated protein (MAP) kinase pathways[32]. Our results showed that the phosphorylation of ERK1/2, one of the major groups of MAP kinase, was

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reduced by miR-34c and SCF siRNA, whereas miR-34c antagomir and forced expression of SCF

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increased the phosphorylation of ERK1/2 in VSMCs.

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Continuous activation of ERK was reported to stimulate cell cycle progression by

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down-regulation of the expression of antiproliferative genes, whereas its inhibition restores those

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genes, resulting in cell cycle arrest in NIH3T3 cells [33]. In our model, a decrease in phosphorylation of ERK by miR-34c mimic was accompanied by an increase in expression Klf4, p21, and p27, all of which are genes related to cell cycle arrest. These observations, coupled with the increase in the expression of pro-apoptotic Bax, suggest that miR-34c interferes with VSMC proliferation and survival partly through the SCF/ERK pathway.

In addition to promoting cell survival [34], AKT is involved in increasing VSMC migration [35] and miR-34c was found to reduce the phosphorylation of AKT and migration in uveal melanoma cells [36]. In cardiac stem cells, migration was shown to be increased by SCF through AKT and MMP-2/9 signaling [37]. On the basis of these previous reports, we speculate that miR-34c decreases VSMC migration by inhibiting SCF.

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The results of the inhibition of either SCF or c-kit by different micro RNAs, including miR-221, miR-222 [38], and miR-494 [2], in different models have been reported. Though these results

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used other cellular models, these signals shared phenomena with our model of VSMCs, such as

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reduced cell survival and migration. These similarities may explain the basis of SCF mediated

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phenotypic modulation.

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In addition to miR-221 and miR-222 both of which target SCF [2, 8], many other miRs have

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been found to regulate neointimal hyperplasia. miR-663, which targets the transcription factor JunB and myosin light chain 9, increases the expression of marker genes of VSMC

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differentiation and inhibits platelet-derived growth factor (PDGF)-induced VSMC proliferation

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and migration [39].Marker genes of VSMC differentiation are also upregulated by miR-145, the

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target of which is KLF5 [7]. Whereas these miRs are protective during vascular remodeling, knocking down miR-21 reduces neointima and lesion formation, which suggests the opposite role in the atherosclerosis [6].

The miR-34 family (miR-34a and b/c) is known to mediate the tumor repressive response of p53. The transcription factor p53 activates the miR-34a and b/c genes in response to cellular stress, including inappropriate oncogene activation [40], and mutation in the p53 pathway is a common feature of various cancers[41]. The tumor-repressive effect of miR-34c includes cell cycle arrest by inhibition of Ccne2, Cdk4, Met, and c-Myc; inhibition of proliferation by inhibition of E2f3 and Myb; and inhibition of migration by inhibition of Met and Cav1 [42]. It was also reported that miR-34c targets sGCβ1, the principal receptor for nitric oxide under hypoxia [10], in pulmonary smooth muscle cells. To our knowledge, however, the effect of miR-34c on VSMCs

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has not previously been reported. Although it is not yet confirmed whether the reduction of neointimal hyperplasia by miR-34c is mediated solely by SCF or by a combination of other

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factors, targeting SCF either with miR-34c or with SCF siRNA is expected to provide useful

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information for treating atherosclerosis.

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5. Conclusions

In the present study, we report that microRNA-34c (miR-34c) targets stem cell factor (SCF) to

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inhibit VSMC proliferation and neointimal hyperplasia. In an animal model, miR-34c was

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significantly increased in the rat carotid artery after catheter injury. Transient transfection of

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miR-34c to either VSMCs or A10 cells inhibited cell survival and migration. miR-34c down-regulated luciferase activity driven by a vector containing the 3’-untranslated region of SCF. Overexpression of SCF in A10 cells induced proliferation and migration, whereas knocking-down of SCF reduced cell survival and migration. miR-34c antagomir-induced VSMC proliferation was blocked by SCF siRNA. Delivery of miR-34c to rat carotid artery attenuated the expression of SCF and blocked neointimal hyperplasia. Thus, we concluded that miR-34c is a novel negative regulator of VSMC proliferation neointima formation by targeting SCF.

Author contributions NC and GHE: performed experiments and analyzed data. J-SK performed surgery. YSK was involved in histology. YKA, YHB, H-YP, and HK interpreted the results of the experiments,

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prepared the figures and drafted the manuscript. HK provided funding, and designed the

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experiments.

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ACKNOWLEDGEMENT

This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of

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Health & Welfare, Republic of Korea (A121260) and the National Research Foundation of Korea grant funded by the Korean government (MEST, #2014-022756)

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REFERENES

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[1] Ambros V, Nature. 2004;431:350-355. [2] Kim WK, Park M, Kim YK, Tae YK, Yang HK, Lee JM, Kim H, Clinical cancer research : an official journal of the American Association for Cancer Research. 2011;17:7584-7594. [3] Lin RJ, Lin YC, Yu AL, Molecular carcinogenesis. 2010;49:719-727. [4] Kim YJ, Hwang SJ, Bae YC, Jung JS, Stem cells. 2009;27:3093-3102. [5] Yoo JK, Jung HY, Lee JM, Yi H, Oh SH, Ko HY, Yoo H, Kim HR, Song H, Kim S, Kim JK, Cell death and differentiation. 2014;21:1150-1159. [6] McDonald RA, White KM, Wu J, Cooley BC, Robertson KE, Halliday CA, McClure JD, Francis S, Lu R, Kennedy S, George SJ, Wan S, van Rooij E, Baker AH, European heart journal. 2013;34:1636-1643. [7] Cheng Y, Liu X, Yang J, Lin Y, Xu DZ, Lu Q, Deitch EA, Huo Y, Delphin ES, Zhang C,

ACCEPTED MANUSCRIPT

Circulation research. 2009;105:158-166. [8] Liu X, Cheng Y, Zhang S, Lin Y, Yang J, Zhang C, Circulation research. 2009;104:476-487.

PT

[9] Kim JR, Kee HJ, Kim JY, Joung H, Nam KI, Eom GH, Choe N, Kim HS, Kim JC, Kook H,

RI

Seo SB, Kook H, The Journal of biological chemistry. 2009;284:16308-16316.

SC

[10] Xu X, Wang S, Liu J, Dou D, Liu L, Chen Z, Ye L, Liu H, He Q, Raj JU, Gao Y, Journal of cell science. 2012;125:6117-6126.

NU

[11] Miyamoto T, Sasaguri Y, Sasaguri T, Azakami S, Yasukawa H, Kato S, Arima N, Sugama

MA

K, Morimatsu M, Atherosclerosis. 1997;129:207-213.

[12] Wang CH, Anderson N, Li SH, Szmitko PE, Cherng WJ, Fedak PW, Fazel S, Li RK, Yau

D

TM, Weisel RD, Stanford WL, Verma S, Circulation research. 2006;99:617-625.

TE

[13] Hollenbeck ST, Sakakibara K, Faries PL, Workhu B, Liu B, Kent KC, The Journal of

AC CE P

surgical research. 2004;120:288-294.

[14] Choe N, Kwon JS, Kim JR, Eom GH, Kim Y, Nam KI, Ahn Y, Kee HJ, Kook H, Atherosclerosis. 2013;229:348-355. [15] de Barros FR, Goissis MD, Caetano HV, Paula-Lopes FF, Peres MA, Assumpcao ME, Visintin JA, Reproduction in domestic animals = Zuchthygiene. 2010;45:38-41. [16] Rodriguez LG, Wu X, Guan JL, Methods in molecular biology. 2005;294:23-29. [17] Owens GK, Kumar MS, Wamhoff BR, Physiological reviews. 2004;84:767-801. [18] Rao RS, Miano JM, Olson EN, Seidel CL, Cardiovascular research. 1997;36:118-126. [19] Takeyoshi M, Yamasaki K, Yakabe Y, Takatsuki M, Kimber I, Toxicology letters. 2001;119:203-208. [20] de Torres C, Munell F, Ferrer I, Reventos J, Macaya A, Neuroscience letters. 1997;230:1-4. [21] Li Q, Dashwood WM, Zhong X, Nakagama H, Dashwood RH, Oncogene.

ACCEPTED MANUSCRIPT

2007;26:6194-6202. [22] Oltvai ZN, Milliman CL, Korsmeyer SJ, Cell. 1993;74:609-619.

PT

[23] Li Z, Zhao J, Li Q, Yang W, Song Q, Li W, Liu J, Cell stress & chaperones.

RI

2010;15:905-912.

SC

[24] Andres V, Urena J, Poch E, Chen D, Goukassian D, Arteriosclerosis, thrombosis, and vascular biology. 2001;21:342-347.

NU

[25] Yang ZY, Simari RD, Perkins ND, San H, Gordon D, Nabel GJ, Nabel EG, Proceedings of

MA

the National Academy of Sciences of the United States of America. 1996;93:7905-7910. [26] Gerthoffer WT, Circulation research. 2007;100:607-621.

D

[27] Wang CH, Verma S, Hsieh IC, Hung A, Cheng TT, Wang SY, Liu YC, Stanford WL,

AC CE P

2007;27:540-547.

TE

Weisel RD, Li RK, Cherng WJ, Arteriosclerosis, thrombosis, and vascular biology.

[28] Zsebo KM, Williams DA, Geissler EN, Broudy VC, Martin FH, Atkins HL, Hsu RY, Birkett NC, Okino KH, Murdock DC, et al., Cell. 1990;63:213-224. [29] Broudy VC, Blood. 1997;90:1345-1364. [30] Wehrle-Haller B, Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society. 2003;16:287-296. [31] Rossi P, Sette C, Dolci S, Geremia R, Journal of endocrinological investigation. 2000;23:609-615. [32] Lennartsson J, Ronnstrand L, Physiological reviews. 2012;92:1619-1649. [33] Yamamoto T, Ebisuya M, Ashida F, Okamoto K, Yonehara S, Nishida E, Current biology : CB. 2006;16:1171-1182. [34] Stabile E, Zhou YF, Saji M, Castagna M, Shou M, Kinnaird TD, Baffour R, Ringel MD,

ACCEPTED MANUSCRIPT

Epstein SE, Fuchs S, Circulation research. 2003;93:1059-1065. [35] Zhang BC, Zhou ZW, Li XK, Xu YW, VASA. Zeitschrift fur Gefasskrankheiten.

PT

2011;40:109-116.

RI

[36] Dong F, Lou D, Molecular vision. 2012;18:537-546.

SC

[37] Guo J, Jie W, Shen Z, Li M, Lan Y, Kong Y, Guo S, Li T, Zheng S, International journal of molecular medicine. 2014;34:112-118.

NU

[38] Poliseno L, Tuccoli A, Mariani L, Evangelista M, Citti L, Woods K, Mercatanti A,

MA

Hammond S, Rainaldi G, Blood. 2006;108:3068-3071.

[39] Li P, Zhu N, Yi B, Wang N, Chen M, You X, Zhao X, Solomides CC, Qin Y, Sun J,

D

Circulation research. 2013;113:1117-1127.

AC CE P

2007;67:8433-8438.

TE

[40] Corney DC, Flesken-Nikitin A, Godwin AK, Wang W, Nikitin AY, Cancer research.

[41] Soussi T, Oncogene. 2007;26:2145-2156. [42] Hermeking H, Cell death and differentiation. 2010;17:193-199.

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Figure Legends

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Fig. 1.Microarray and quantitative real-time RT-PCR analysis revealed that the expression of

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miR-34c-5p was increased in injured carotid artery. (A) MicroRNA-34c stem loop sequence. (B)

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Time course of the changes in miR-34c expression 1, 7, and 14 days after balloon injury. The

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increase in expression of miR-34c-5p was observed 7 days after injury.

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Fig. 2.miR-34c attenuated VSMC proliferation and migration. (A) MTT assay revealed the negative effect of miR-34c on survival of A10 cells, a rat vascular smooth muscle cell (VSMC)

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line. (B-D) The result of MTT assay (B) and direct counting (C and D) of VSMCs also indicated

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an anti–proliferative effect of miR-34c. (E) Results of BrdU(upper panels) and TUNEL (lower

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panels) assay. Transient transfection of miR-34c mimic decreased BrDU uptake and increased apoptosis, whereas the antagomir of miR-34c had the opposite effects. (F) Expression of the anti-apoptotic protein Bcl2 and of negative regulators of the cell cycle including p21, p27, and Klf4 was increased, whereas that of the pro-apoptotic protein Bax was down-regulated by miR-34c. The Western blot data were normalized by Gapdh.

Fig. 3.miR-34c attenuated cell migration, whereas miR-34c antagomir potentiated cellular movement. (A) The wound healing assay was performed. (B) Quantification results of the wound healing assay. (C) Transwell migration assay. (D) Quantification results.

Fig. 4.miR-34c targets SCF. (A) Sequence alignment between miR-34c-5p and the 3’-UTR of SCF mRNA. (B) Transfection of miR-34c mimic reduced transcription of SCF in A10 cells. (C)

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Expression of the luciferase gene with the 3’-UTR sequence of SCF was down-regulated by

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miR-34c in a dose-dependent fashion.

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Fig. 5. SCF induces VSMC proliferation and migration. (A-B) Overexpression of SCF increased

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cell proliferation (A), whereas knocking down of SCF with siRNA decreased cell survival (B). (C-D) The wound healing assay (C) and Transwell migration assay (D) revealed that inhibition

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of SCF attenuated cell migration. (E-F) Overexpression of SCF increased protein expression of

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Bcl2 and phosphorylation of Akt and Erk (E), whereas knocking down of Scf had the opposite

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effect (F).

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Fig. 6.The miR-34c antagomir-induced increases in proliferation and migration were blocked by

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knocking-down of SCF. The results of direct counting (A) and the Transwell migration assay (B) were performed.

Fig. 7. Changes in the expression of SCF in carotid artery injury model. (A) Immunohistochemical analysis against SCF showed that the expression of SCF was increased 7 days after injury and then lowered at 14 days. (B) Quantification of SCF positive tissues of Fig. 7A. Immunopositivity was measured as described in the methods section from 4 independent sets of experiments. (C) Quantitative real time RT-PCR results. Compared with the contralateral carotid artery, transcription of SCF was down-regulated 1 day after injury. However, the mRNA level was significantly increased 7 days after injury compared with injured artery at 1 day, which was then normalized at day 14.

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Fig. 8. Local delivery of miR-34c attenuated atherosclerosis in rat carotid artery model. (A) H&E staining of rat carotid artery after balloon injury. Local delivery of miR-34c significantly reduced

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the neointimal hyperplasia. (B)Luminal area. (C)Neointimal area. (D) Neointima to media ratio.

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(E) Percentage stenosis (F). Immunohistochemical analysis showed that the expression of SCF

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Highlights

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 miR-34c was significantly increased in the rat carotid artery 14 days after injury.

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 miR-34c inhibited vascular smooth muscle cell survival and migration.

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 miR-34c targets SCF.

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 Knocking-down of SCF by miR-34c reduced A10 cell survival and migration.

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 miR-34c attenuated the expression of SCF and neointimal hyperplasia in rat artery.