Protein and miRNA profiling of radiation-induced skin injury in rats: the protective role of peroxiredoxin-6 against ionizing radiation

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Protein and miRNA profiling of radiationinduced skin injury in rats: the protective role of peroxiredoxin-6 (PRDX-6) against ionizing radiation Shuyu Zhang, Wenjie Wang, Qing Gu, Jiao Xue, Han Cao, Yiting Tang, Xiaohui Xu, Jianping Cao, Jundong Zhou, Jinchang Wu, Wei-Qun Ding www.elsevier.com/locate/freeradbiomed

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S0891-5849(14)00032-X http://dx.doi.org/10.1016/j.freeradbiomed.2014.01.019 FRB11879

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Free Radical Biology and Medicine

Received date: 16 August 2013 Revised date: 8 January 2014 Accepted date: 13 January 2014 Cite this article as: Shuyu Zhang, Wenjie Wang, Qing Gu, Jiao Xue, Han Cao, Yiting Tang, Xiaohui Xu, Jianping Cao, Jundong Zhou, Jinchang Wu, Wei-Qun Ding, Protein and miRNA profiling of radiation-induced skin injury in rats: the protective role of peroxiredoxin-6 (PRDX-6) against ionizing radiation, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2014.01.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title page

Protein and miRNA profiling of radiation-induced skin injury in rats: the protective role of peroxiredoxin-6 (PRDX-6) against ionizing radiation

Shuyu Zhanga,*, Wenjie Wangb, Qing Gua, Jiao Xuea, Han Caoa, Yiting Tanga, Xiaohui Xuc, Jianping Caoa,*, Jundong Zhoud, Jinchang Wud, Wei-Qun Dinge

a

School of Radiation Medicine and Protection and Jiangsu Provincial Key Laboratory of

Radiation Medicine and Protection, Soochow University, Suzhou 215123, China b

Cyrus Tang Hematology Center, Soochow University, Suzhou 215123, China

c

Deaprtment of General Surgery, the Second Affiliated Hospital of Soochow University,

Suzhou 215004, China d

Department of Radio-Oncology, Nanjing Medical University Affiliated Suzhou Hospital,

Suzhou 215001, China e

Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma

City 73104, United States

*Corresponding authors: (S. Zhang) No. 199 Ren’ai Rd, Suzhou 215123, China. Tel/Fax: +86512-65880065, E-mail: [email protected]; (J. Cao) No. 199 Ren’ai Rd, Suzhou 215123, China. Tel/Fax: +86512-65880037, E-mail: [email protected]

The first two authors contributed equally to this work.

1

Abstract Radiation-induced skin injury is a serious concern during radiotherapy. However, the molecular mechanism underlying the pathogenesis of radiation-induced skin injury has not been extensively reported. Most biological functions are performed and regulated by proteins and non-coding RNAs, including microRNAs (miRNAs). The interplay between mRNA and miRNA has been implicated in disease initiation and progression. Technical advances in genomics and proteomics have enabled the exploration of the etiology of diseases and have the potential to broaden our understanding of the molecular pathogenesis of radiation-induced skin injury. In this study, we compared the protein and miRNA expression in rat skin irradiated with a 45 Gy electron beam with expression from adjacent normal tissues. We found 24 preferentially expressed proteins and 12 dysregulated miRNAs in irradiated skin. By analyzing the protein and miRNA profiles using bioinformatics tools, we identified a possible interaction between miR-214 and peroxiredoxin-6 (PRDX-6). Next, we investigated the expression of PRDX-6 and the consequences of its dysregulation. PRDX-6 is suppressed by radiation-inducible miR-214 and is involved in the pathogenesis of radiation-induced skin injury. Overexpression of PRDX-6 conferred cells radioresistance, decreased cell apoptosis and preserved mitochondrial integrity after radiation exposure. In addition, in vivo transfection with PRDX-6

reduced

radiation-induced

reactive

oxygen

species

(ROS)

and

the

malondialdehyde (MDA) concentration and ameliorated radiation-induced skin damage in rats. Our present findings illustrate the molecular changes during radiation-induced skin injury and the important role of PRDX-6 in ameliorating this damage in rats.

Key words: radiation-induced skin injury; protein and miRNA profiling; peroxiredoxin-6 (PRDX-6)

2

Introduction Ionizing radiation is used extensively to treat many different types of cancer and contributes to 40% of curative cancer treatments [1,2]. However, radiotherapy may significantly injure the skin and profoundly impair its function [3,4]. Radiation-induced skin injury is a serious concern that may limit the duration and the delivered dose of radiation. In addition, the increasing use of radioactive materials in industry, medicine, science, the military and nuclear facilities with localized areas of high radiation has significantly increased the potential of large-scale, uncontrolled exposure to radiation [5,6]. During radiation exposure, skin tissue damage occurs instantaneously and is mediated by a burst of free radicals. Irradiated cells produce reactive oxygen and nitrogen species (ROS/RNS), including hydroxyl radicals, superoxide anion, hydrogen peroxide and nitrogen dioxide. The detrimental ROS can result in damage to nuclear DNA and alteration of proteins, lipids and carbohydrates, leading to changes in the cell network [7,8]. Pathophysiological changes in radiation-induced skin damage include erythema and desquamation, which occur within hours or weeks, as well as dermal atrophy and telangiectasia, which occur over the long term [9]. Most biological functions are performed and regulated by proteins and non-coding RNAs, including microRNAs (miRNAs). Previous studies have demonstrated that numerous proinflammatory cytokines and chemokines are involved in radiation-induced skin injury, including tumor necrosis factor- (TNF-), transforming growth factor- (TGF-) and CXC chemokine ligand 12 (CXCL-12) [10-12]. However, reports describing radiation-induced skin injury are limited. Systematic analysis of the whole proteome may extend our understanding of the molecular pathogenesis of this injury. It may also provide precious comprehensive information about the molecular basis of radiation-induced skin injury and the course of the damage. miRNAs are small, non-coding regulatory RNAs that suppress gene expression through partial complementary elements in the 3’UTRs of their target mRNAs [13,14]. Most animal miRNAs are evolutionarily conserved and occur in clusters [15]. After transcription and processing, mature miRNAs are assembled into a miRNA-induced silencing complex, which directs binding to the cognate sequence in the 3’ UTR of target mRNAs. miRNAs 3

are implicated in a wide variety of biological processes, including cell proliferation, apoptosis, metabolism, cell differentiation and disease initiation and promotion [16]. Identification of the miRNAs involved in radiation-induced skin damage may provide new insights into its progression [17]. The protein and miRNA profiles of radiation-induced skin injury have not been analyzed, and there is currently little information published on the mechanism of damage. Extensive investigation of the molecular etiology as well as the treatment of radiation-induced skin injury is warranted. In this study, we investigated the protein and miRNA profiles of irradiated skin. Over 20 preferentially expressed proteins and 12 dysregulated miRNAs were identified. We further investigated the mechanism of decreased PRDX-6 and the consequence of its dysregulation. PRDX-6, which is regulated by miR-214, was involved in the pathogenesis of radiation-induced skin injury. Overexpression of PRDX-6 conferred radioresistance to cells and decreased cell apoptosis after radiation exposure. In addition, in vivo transfection with PRDX-6 ameliorated radiation-induced damage to rat skin. These results suggest that PRDX-6 has a protective role against radiation-induced skin injury.

Materials and methods Animals and treatments Male SD rats (4 weeks old) were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Rats were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg), and the hair on the rat buttock was shaved using a razor. Rats were immobilized with adhesive tape on a plastic plate to minimize motion during radiation exposure. A 3-cm-thick piece of lead was used to shield the rats and localize the radiation field (3 cm × 4 cm). A single dose of 45 Gy irradiation was administered to the treatment area at a dose rate of 750 cGy/min using a 6-MeV electron beam accelerator (Clinac 2100EX, Varian Medical Systems, Inc., CA). This dose was selected because it can significantly induce skin injury (100% of animals develop grade 4-5 injury at this dose) [18]. Three days after irradiation, skin tissues were subjected to protein and miRNA profiling analysis. For animal studies, rats randomly received one of the following treatments: 1) 200-l 4

subcutaneous injection of PBS, 2) subcutaneous transfection with 2 g pcDNA3.1 vector using in vivo-jetPEI delivery reagent (Polyplus, Illkirch, France) and 3) subcutaneous transfection with 2 g pcDNA3.1-PRDX6 vector. Skin reactions were graded at regular intervals from 1 (no damage) to 5 (severe damage) using the semi-quantitative skin injury scale as previously described [18,19]. All the protocols and procedures were approved by the Animal Experimentation Ethics Committee of the Soochow University. 2D electrophoresis (2-DE) Selected samples were subjected to 2D gel electrophoresis using IPG strips (3-10 pH range, Bio-Rad Laboratories, Hercules, CA). Samples containing up to 100 g protein were diluted to 320 L with rehydration solution (7 mol/L urea, 2 mol/L thiourea, 2% CHAPS, 100 mmol/L DTT, 0.2% w/v Bio-Lyte of pH 3-10, and trace bromophenol blue) and rehydrated into IPG strips overnight. Isoelectric focusing (IEF) was performed at 34 000 Vh using a PROTEAN IEF system (Bio-Rad). The IPG strips were then soaked in equilibration buffer (50 mmol/L Tris-HCl of pH 8.8, 6 mol/L urea, 30% glycerol, 2% SDS and 0.001% bromophenol blue). Next, they were soaked in equilibration buffer containing 45 mg/mL iodoacetamide for 15 min. Equilibrated IPG strips were transferred onto 12% uniform polyacrylamide gels and were run for 5 h. The gels were visualized using the silver staining method. After staining, 2-DE gels were scanned using the Fluor-STM MultiImager (Bio-Rad), and images were analyzed using PDQuest (Bio-Rad). Protein identification To accurately compare the spots between gels, image spot intensity was normalized by dividing the raw intensity of each spot by the total intensity of all the valid spots in a gel. Protein spots of interest were excised from the polyacrylamide gels using a robotic workstation (Investigatore Propice, Genomics Solutions, Ann Arbor, MI) and were trypsin-digested using a robotic digestion system (ProGeste, Genomic Solutions). Finally, peptides were analyzed on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA). Mass spectrometry data were searched against the rat protein database of the SWISS-PROT database. miRNA microarray 5

Total RNA was extracted from cells or tissues with TRIzol reagent (Invitrogen, Carlsbad, CA). Microarray-based miRNA expression profiling was performed using the miRCURY LNA rat microRNA Array (Exiqon, Vedbaek, Denmark). The microarrays contained approximately 1200 assay probes corresponding to all of the annotated rat miRNA sequences (miRBase, version 12, 2008; the Wellcome Trust Sanger Institute, Cambridgeshire, UK). Total RNA labeling and hybridization were performed using standard conditions according to the manufacturer’s instructions. RNA extraction and real-time PCR Total RNA from skin tissues was extracted with TRIzol (Invitrogen, Carlsbad, CA). The mRNAs were reverse transcribed into cDNA using an oligo(dT)12 primer and Superscript II reverse transcriptase (Invitrogen). SYBR green dye (Takara Bio Inc., Shiga, Japan) was used for amplification of cDNA. The levels of ATP synthase  subunit, elongation factor-1, heat shock protein 1, calcium-binding protein 1, PRDX-6 and the internal standard -Actin mRNA were measured by quantitative real-time PCR in triplicate. The primer sequences are listed in Supplementary Table 1. The expression levels of mature miRNAs were quantified by real-time PCR according to the manufacturer’s protocols (Genepharma, Shanghai, China). Briefly, 5 ng of total RNA were reverse transcribed using specific stem-loop RT primers, after which they were amplified and detected using PCR with specific primer probes. U6 snRNA (RNU6B) served as an internal normalized reference. Thermocycler conditions included an initial step at 95°C for 2 min followed by 35 cycles at 95°C for 15 sec and 55°C for 1 min. The quantitative real-time PCR results were analyzed and expressed as relative miRNA or mRNA levels based on the Ct (cycle threshold) value and then converted to fold change. Skin cell isolation and culture Human keratinocyte HaCaT cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and antibiotics (100 units/ml penicillin G and 100 units/ml streptomycin sulfate; Gibco, Grand Island, NY). Primary rat skin cells were isolated from adult (5–6 weeks old) rat skin using the procedures described previously [20,21]. Vector construction, cell transfection and luciferase assays 6

Luciferase vectors with full-length PRDX-6 3’UTR (pGL3-PRDX6-UTR) were constructed by Shanghai Biobuy Biotech Co. Ltd. (Shanghai, China). The plasmids were confirmed by sequencing. Chemically synthesized RNAs, including negative control (NC) RNA, rat miR-214 mimics and rat miR-214 inhibitor were obtained from GenePharma (GenePharma, Shanghai, China). Cells were transfected with constructed vectors or synthetic RNAs by Lipofectamine 2000 (Invitrogen). In each transfection, 50 ng of pRL-TK (Promega, Madison, WI) was used to correct for the transfection efficiency. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega). Promoter activity was expressed as the ratio of Firefly luciferase to Renilla luciferase activity. Clonogenic assay For standard clonogenic assays, cells were transfected with the PRDX-6 expression vector or control vector. Twenty-four hours after the transfection, the cells were seeded in 6-well plates at 200-1,000 cells/well, depending on the dose of radiation. The cells were irradiated using a 6-MV X-ray linear accelerator (Clinac 2100EX; Varian Medical Systems, Inc., CA) at a dose rate of 2 Gy/min; a 1.5-cm bolus was used as a compensator. After radiation, the medium was immediately replaced with fresh DMEM medium. The cells were then grown for 7-10 days to allow for colony formation, fixed and stained using crystal violet. Colonies consisting of 50 or more cells were considered clones. MitochondrialLQWHJULW\DVVD\ Mitochondrial integrity was evaluated using Mito-Tracker Red (Invitrogen). Cells were transfected with vectors 24 h prior to receiving 20 Gy of X-ray irradiation. Twenty-four hours after irradiation, the cells were incubated for 30 min in the dark with Mito-Tracker Red dissolved in serum-free medium at 37°C. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO). ROS generation assay ROS levels were determined using the ROS-sensitive dye 2’,7’-dichlorofluorescein diacetate (DCF-DA), which is converted by ROS into the highly fluorescent 2’,7’-dichlorofluorescein (DCF). HaCaT cells were washed with phosphate buffer (pH 7.4) 7

and incubated with DCF-DA (10 M) for 30 min. The free radical scavenger N-acetylcysteine (NAC, Sigma-Aldrich, St. Louis, MO) and superoxide dismutase 1 (SOD1) overexpression vector were used as positive controls. The level of DCF fluorescence, reflecting the concentration of ROS, was measured by a fluorescence microscope. For skin tissues, the level of DCF fluorescence was measured at 488 nm using a 96-well plate reader. Measurement of apoptosis Cells were transfected with the PRDX-6 expression vector or control vector 24 h prior to receiving 20 Gy of irradiation. Apoptosis was measured using propidium iodide (PI)/annexin-V double staining (Beyotime, Nantong, China). The cells were harvested 48 h after irradiation with 20 Gy radiation. Apoptotic fractions were measured using flow cytometry (Beckman, CA). Malondialdehyde (MDA) concentration measurement Tissue MDA levels were determined using the thiobarbituric acid (TBA) assay as described previously [19]. MDA levels were expressed as nM/mg protein. Western blot Cells were transfected with RNAs 24 h prior to irradiation with 20 Gy of radiation. The cells were then washed twice with ice-cold PBS and directly lysed in 200 μl of cell lysis buffer. The lysates were boiled, centrifuged at 10,000 rpm, and then loaded onto a 12% SDS-PAGE gel. The samples were electrophoresed for 2 h and transferred onto transfer membranes. After blocking with 5% non-fat milk in PBS-Tween 20 for 1 h at room temperature, the membranes were blotted with PRDX-6 or GAPDH primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1,000 dilutions. The membranes were then incubated with the appropriate horseradish peroxidase-coupled secondary antibody at a 1:2,000 dilution for 1 h at room temperature. After the membranes were washed with TBST, the blots were incubated in detection reagent (Amersham Biosciences, Freiburg, Germany) and exposed to Hyperfilm ECL film (Pierce, Rockford, IL). Hematoxylin and eosin (H&E) staining Skin tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Three-micrometer paraffin sections were deparaffinized and heat-treated with citrate 8

buffer of pH 6.0 for 7 min following an epitope retrieval protocol. The sections of rat skin were stained with H&E. Statistical analysis All analyses represent experiments that were performed at least in triplicate. The results were evaluated by one- or two-way ANOVA to determine statistical significance. The radiation sensitivity enhancement ratio (SER) was measured according to the multi-target single hit model. For ordinal data, the Mann–Whitney test was used. Statistical analysis was performed using SPSS software. Data were considered significant if P < 0.05.

Results Protein identification by 2-DE and MS Proteins from skin tissues irradiated with 45 Gy of radiation and nonirradiated control skin tissues were separated with pH 3-10 IPG strips. High resolution and reproducible 2-DE profiles were obtained (Fig. 1A and 1B). Thirty protein spots were excised from the silver staining gel and digested in the gel with trypsin. Of these, 24 protein spots were identified successfully. Among the identified proteins (Table 1), there were three overexpressed and 21 underexpressed in irradiated skin tissues compared with corresponding normal skin tissues. The identified differentially expressed proteins included mitochondrial aspartate

aminotransferase, elongation factor-1, 14-3-3 protein , peroxiredoxin-6 (PRDX-6, Fig. 1C) and ATP synthase  subunit. To further confirm the expression of dysregulated molecules, we expanded our sample to include six pairs of skin specimens. Real-time PCR analysis showed that the levels of ATP synthase  subunit, elongation factor-1, heat shock protein 1 and PRDX-6 mRNA were significantly dysregulated in irradiated skin compared with matched nonirradiated tissues (P < 0.05; Fig. 1D-1G), However, the levels of calcium-binding protein 1 mRNA was not significant different between the two groups (Fig. 1H). The results suggest that these molecules may be novel factors associated with the pathogenesis of radiation-induced skin damage. miRNAs are dysregulated in radiation-induced rat skin To search for miRNAs that may be involved in the progression of radiation-induced skin damage, irradiated rat skin and control tissues were screened for differentially expressed 9

miRNAs by miRNA-array analysis. The raw array measures are accessible in Supplementary Table 2. We found that many miRNAs were significantly dysregulated in irradiated skin tissues, including miR-223, miR-21, miR-34a, and miR-214 (Fig. 2A and Table 2). To further confirm the expression of the dysregulated miRNAs, we expanded our sample to include an additional six pairs of skin specimens. Real-time PCR analysis showed that miR-223, miR-21, miR-17-5p and miR-214, but not miR-301a, were significantly dysregulated in irradiated skin compared with matched nonirradiated tissues (P < 0.05; Fig. 2B-2F). This suggests that these miRNAs may be involved in radiation-induced skin damage.

Peroxiredoxin-6 (PRDX-6) is a direct target of miR-214 Bioinformatics algorithms (TargetScan and miRanda) were used to explore the possible interactions between miRNAs and proteins that had been altered by radiation. These tools consistently suggest that miR-214 may directly regulate rat PRDX-6. One binding site for miR-214 was observed at position 196-230 in the 3‘UTR of PRDX-6 mRNA (Fig. 3A). To test the hypothesis that PRDX-6 is a target of miR-214, we constructed a reporter by inserting the wild-type fragments from the 3‘UTR region of PRDX-6 downstream of the luciferase coding region (pGL3-PRDX6-UTR). Oligonucleotides of miR-214 mimics or control miRNA were cotransfected with these reporter plasmids into human HaCaT cells. The luciferase assay showed that miR-214 mimics repressed the activity of pGL3-PRDX6-UTR markedly (Fig. 3B). Similar results were observed using primary skin cells from rats (Fig. 3C). Western blot analysis indicated that the level of PRDX-6 protein was reduced significantly by miR-214 overexpression compared with control miRNA (miRNA-NC, Fig. 3D), suggesting that PRDX-6 is a direct target of miR-214. The effect of PRDX-6 on the radiosensitivity of HaCaT cells To investigate the effect of PRDX-6 on the radiosensitivity of HaCaT cells, we performed an in vitro clonogenic cell survival assay using PRDX-6 treatment plus radiation. HaCaT cells transfected with PRDX-6 and irradiated with X-rays exhibited significantly higher clonogenic survival rates than cells treated with radiation alone. Compared with pcDNA3.1-transfected cells, the radiation sensitivity enhancement ratio (SER) was 0.83 for cells transfected with PRDX-6 (Fig. 4A). The data were further analyzed using two-way 10

ANOVA to test the relationship between PRDX-6 and radiation. Our results indicated that the interaction between PRDX-6 transfection and radiation was statistically significant (P = 0.003) for HaCaT cells. These results demonstrate that transfection with PRDX-6 could attenuate the radiosensitivity of human skin cells. The inhibition of ROS, mitochondrial damage and apoptosis by PRDX-6 Ionizing radiation elicits cutaneous free radical reactions. We therefore examined the levels of ROS in HaCaT cells using DCF-DA assay. Results demonstrated that ROS level was significantly increased 24 h after irradiation, whereas PRDX-6-transfected cells showed

significantly

reduced

ROS

level,

compared

with

control

group

or

pcDNA3.1-transfected group (Fig. 4B and 4C). The rigor of PRDX-6 in ROS scavenging was similar to that of the well-established SOD1 and NAC in HaCaT cells. Mitochondrial functional failure is considered to be one of the most important factors causing cell death [22]. Ionizing radiation may disrupt mitochondrial structure and function. Damage to mitochondrial membrane permeability and membrane potential changes that are caused by free radicals contribute to cell apoptosis [23-24]. To explore the protective role of PRDX-6 on mitochondrial function, Mito-tracker Red staining was used. As shown in Fig. 4D and 4E, nonirradiated HaCaT cells had brilliant staining of Mito-tracker Red, which was attenuated and morphologically changed by 20 Gy of irradiation. Comparatively, PRDX-6-transfected cells had significant stronger staining, indicating the integrity of the mitochondrial membrane is maintained after ionizing radiation and PRDX-6 protects the mitochondrion against ionizing radiation. We next investigated whether the increased clonogenic survival by treatment with PRDX-6 was associated with decreased apoptosis. PI/Annexin-V staining-based flow cytometric analysis of apoptosis was performed. The proportion of apoptotic cells is shown in Fig. 4F. PRDX-6 treatment reduced the apoptosis of HaCaT cells after 20 Gy of irradiation relative to cells transfected with a control vector (16.29% vs. 9.21%, P = 0.003). These results demonstrate that PRDX-6 reduces the apoptotic cell death of HaCaT cells caused by irradiation. The inhibition of ROS and mitochondrial damage by miR-214 inhibitors Because radiation-induced increase in miR-214 level led to a suppression of PRDX-6 11

expression, we hypothesize that inhibition of miR-214 could protect skin cells against radiation-induced damage. Results showed that transfection of miR-214 inhibitor significantly decreased ROS level, compared with mock transfected or miRNA inhibitor negative control transfected cells (Fig. 5A and 5B). Moreover, transfection of miR-214 inhibitor also showed increased mitochondrial integrity after 20 Gy irradiation (Fig. 5C and 5D). These results indicated that inhibition of miR-214 protected cells against irradiation, which mimicked the effect of PRDX-6 overexpression. PRDX-6 overexpression ameliorates radiation-induced skin injury To determine whether PRDX-6 overexpression by in vivo transfection could attenuate radiation-induced skin injury, we performed an experiment using a single dose of 45 Gy irradiation delivered to the buttock skin of SD rats followed by a subcutaneous injection of control vector (pcDNA3.1) or PRDX-6 expression vector. The rats in the control group were injected with an equivalent volume of PBS. Twenty-four hours after irradiation, the ROS levels in the skin tissues were measured. The 45 Gy irradiation significantly increased skin ROS levels, while transfection with PRDX-6, but not pcDNA3.1, reduced the generated ROS levels (Fig. 6A). Because radiation-induced ROS result in oxidative damage to lipids, DNA and proteins, cellular defenses are proposed to play important roles in protecting skin cells against oxidative stress and in reducing the progression of skin injury [22,23]. To test whether overexpression of PRDX-6 affects radiation-induced lipid peroxidation, the concentration of MDA in skin tissues after 45 Gy irradiation was measured. As shown in Fig. 6B, MDA levels were significantly decreased in tissues transfected with pcDNA3.1-PRDX6 compared with the control group (P < 0.05), whereas transfection with the pcDNA3.1 empty vector did not reduce the production of MDA. This indicated that PRDX-6 overexpression attenuates lipid peroxidation resulted from radiation-induced oxidation or/and amplification by autoxidation. Injuries to skin tissues were graded on a scale of 1 (no damage) to 5 (severe damage) as described previously [18,19]. Cutaneous damage was observed 5-6 days after irradiation. Skin injury reached a maximum at 18-27 days after irradiation, after which the wounds began to heal. Radiation-induced skin injury was significantly less severe in the 12

PRDX-6 overexpression group compared with PBS-treated rats beginning 24 days after irradiation, while rats receiving pcDNA3.1 had skin tissue damage that was similar to that of the control group (Fig. 7A and 7B). Although transfection with PRDX-6 showed similar skin surface damage with mock-treated or pcDNA3,1-tranfected rats 60 days post irradiation (Fig. 7A and Fig. 7C), PRDX-6 overexpression attenuated epidermal hyperplasia (white arrow) and maintained skin appendages (blue arrow), which were destroyed by irradiation (Fig. 7D and 7E).

Discussion Radiation-induced skin injury caused by radiotherapy is a serious concern, and its molecular pathogenesis remains elusive. This is the first report to describe the changes in protein and miRNA expression in response to electron beam irradiation, providing insight into the molecular pathogenesis of radiation-induced skin injury. After comprehensive interrogation of the proteome and transcriptome, we identified novel molecules implicated in radiation-induced skin injury (Table 1 and 2). For example, elongation factor-1 is implicated in stress-induced apoptosis [25]. The creatine kinase system has exhibited protective effects against oxidative- and UV-induced skin damage [26]. The decline in creatine kinase expression may attenuate the energy supply in radiation-treated skin, causing detrimental changes in cutaneous energy metabolism. Protein disulfide isomerase is induced by stress and is protective against apoptotic cell death [27]. Using miRNA-array analysis of irradiated rat skin, we found that 8 miRNAs were overexpressed and 4 were underexpressed in radiation-induced skin tissues compared with adjacent nonirradiated tissues. Some of these differentially expressed miRNAs have been identified in previous studies. Notably, miR-21 is stimulated by ionizing radiation in different types of mammalian cells [28,29]. miR-34a is a radiation-responsive miRNA induced in a p53-dependent manner in multiple organs of mice [30,31]. miRNAs exert their functions through mRNA decay or translation inhibition [13,14]. In the latter mechanism, the mRNA level of a protein-coding gene usually remains unchanged, while protein level is affected. Unlike most studies that perform correlation analysis by miRNA and mRNA profiling, here we analyzed the relationships between the miRNA and protein 13

profiles and identified PRDX-6 as a direct miR-214 target. Radiation-induced oxidative stress produces ROS that damage DNA and most other biological macromolecules. Oxidative stress also conveys signals to the cell nucleus to induce stress responses such as cell-growth arrest and apoptosis. To prevent such cellular damage, living organisms have evolved a defense system involving several antioxidant enzymes as well as antioxidant molecules [32,33]. These antioxidant enzymes such as SOD1, SOD2, catalase, and glutathione peroxidases (GPxs) contribute to cellular defense against irradiation [34-36].

The results from the present study indicate that

miR-214/PRDX-6 interaction is a novel mechanism by which we may protect radiation-induced skin injury. The proportional contribution of individual antioxidant systems in protecting against radiation-induced skin damage merits further investigation. The peroxiredoxin (PRDX) family consists of several peroxidases that detoxify hydrogen peroxide and various organic peroxides. All PRDX proteins contain a conserved cysteine residue, cysteine 47. PRDX-1 to -5 contain two reactive cysteines and use thioredoxin and/or glutathione as a substrate, whereas PRDX-6 has a single redox-active cysteine and uses glutathione to catalyze the reduction of H2O2 and various organic peroxides. The expression of PRDX-6 is strongly increased in the hyperproliferative epidermis of wounded and psoriatic skin, suggesting that this enzyme has a role in skin homeostasis [37,38]. PRDX-6 overexpression strongly decreases apoptosis induced by ROS in vitro and in vivo [39], suggesting that activation of PRDX-6 could be a novel strategy for skin protection under stress conditions. Moreover, PRDX-6 level may also inversely associated with natural oxidation damage to the skin. Considering the bioavailability of plasmid transfection, virus- or protein transduction domain (PTD)-mediated PRDX-6 delivery may be potentially therapeutic protective. -

-

A variety of ROS, such as •OH, O2• , •O2H, ONOO , and H2O2, are generated upon ionizing radiation exposure. These radicals together with secondary radicals derived from -

ONOO in the superoxide-generating environment are likely to cause oxidative damage to lipids, DNA and proteins [40]. Skin is rich in lipids, proteins and DNA, which makes it one of the most sensitive and targeted organs for oxidative stress [41]. Hydrogen peroxide is a strong oxidizer, and endogenous catalase and peroxidases can harmlessly and 14

catalytically decompose low concentrations of hydrogen peroxide to water and oxygen. Hydroperoxides produced by ionizing radiation and subsequent metabolic processes may contribute to persistent ionizing radiation-induced genomic instability [42]. Ionizing radiation-induced genomically unstable cells have a 3-fold increase in steady-state levels of hydrogen peroxide but no increase in superoxide [42]. In the present study, we demonstrated that radiation induced the expression of miR-214, which suppressed the protective antioxidant PRDX-6 in rat skin. Forced expression of PRDX-6 reduced radiation-induced ROS, maintained the integrity of the mitochondrion and reduced cell apoptosis, indicating that PRDX-6 attenuates radiation-induced free radicals. This suggests that PRDX-6 enhances the antioxidative responses of skin cells to radiation insults and attenuates the severity of skin injury. However, the overexpression of PRDX-6 should be restricted to normal tissues. Inappropriate delivery of PRDX-6 into tumor cells may result in their resistance to ionizing radiation. Because oxidative stress has a critical role in the progression of radiation-induced skin injury, supplementation of antioxidant enzymes or compounds has been utilized to mitigate this injury. It is well known that detrimental superoxide anion, generated during Ionizing radiation, can be converted to hydrogen peroxide by SOD. Yan et al. mitigated radiation-induced skin injury with AAV-mediated MnSOD (SOD2) expression in a mouse model [43]. MnSOD has also been shown to decrease superoxide levels and protect the bladder from radiation damage [44]. A recent study reports the mitigation of radiation dermatitis and promotion of wound healing using synthetic superoxide dismutase/catalase mimetic EUK-207 [45]. We have previously reported that overexpression of antioxidant heme oxygenase-1 (HO-1) and SOD1 ameliorates radiation-induced skin injury [18,46]. Our present results indicate PRDX-6 as an important novel antioxidant enzyme for the progression and treatment of radiation-induced injury. Because radiation generates multiple types of ROS, exploring whether a combination of antioxidant enzymes could be more effective in reducing radiation-induced skin damage is warranted.

Conclusions In summary, we found over 20 preferentially expressed proteins and 12 dysregulated 15

miRNAs in radiation-induced skin tissues. We investigated the underlying mechanism of decreased PRDX-6 and the consequence of its dysregulation. PRDX-6, which is regulated by miR-214, was involved in the pathogenesis of radiation-induced skin injury. Overexpression of PRDX-6 conferred cell resistance to radiation and decreased cell apoptosis after radiation. In addition, in vivo transfection with PRDX-6 ameliorated radiation-induced skin damage in rats. These results suggest that PRDX-6 has a protective role against radiation-induced skin injury.

Conflict of Interest Statement: None declared.

Acknowledgements This work is supported by the National Natural Science Foundation of China (81102078, 81172597 and 81372433) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

References [1] Begg AC, Stewart FA, Vens C. Strategies to improve radiotherapy with targeted drugs. Nat Rev Cancer 11:239-253; 2011. [2] Baskar R, Lee KA, Yeo R, Yeoh KW. Cancer and radiation therapy: current advances and future directions. Int J Med Sci 9:193-199; 2012. [3] Hymes SR, Strom EA, Fife C: Radiation dermatitis. clinical presentation, pathophysiology, and treatment 2006. J A m Acad Dermatol 54:28-46; 2006. [4] Yi A, Kim HH, Shin HJ, Huh MO, Ahn SD, Seo BK. Radiation-induced complications after breast cancer radiation therapy: a pictorial review of multimodality imaging findings. Korean J Radiol 10:496-507; 2009. [5] Gottlober P, Bezold G, Weber L, Gourmelon P, Cosset JM, Bahren W, et al. The radiation accident in Georgia: clinical appearance and diagnosis of cutaneous radiation syndrome. J Am Acad Dermatol 42:453-458; 2000. 16

[6] Balli E, Comelekoglu U, Yalin E, Yilmaz N, Aslantas S, Sogut F, et al. Exposure to gamma rays induces early alterations in skin in rodents: Mechanical, biochemical and structural responses. Ecotoxicol Environ Saf 72:889-894; 2009. [7] Spitz DR, Azzam EI, Li JJ, Gius D. Metabolic oxidation/reduction reactions and cellular responses to ionizing radiation: a unifying concept in stress response biology. Cancer Metastasis Rev 23:311-322; 2004. [8] Watters D. Molecular mechanisms of ionizing radiation-induced apoptosis. Immunol Cell Biol 77:263-271; 1999. [9] Chi C, Tanaka R, Okuda Y, Ikota N, Yamamoto H, Urano S, et al. Quantitative measurements of oxidative stress in mouse skin induced by X-ray irradiation. Chem Pharm Bull (Tokyo) 53:1411-1415; 2005. [10] Martin M, Lefaix J, Delanian S. TGF-beta1 and radiation fibrosis: a master switch and a specific therapeutic target? Int J Radiat Oncol Biol Phys 47:277-290; 2000. [11] Kim JH, Kolozsvary AJ, Jenrow KA, Brown SL. Mechanisms of radiation-induced skin injury and implications for future clinical trials. Int J Radiat Biol 2013. [Epub ahead of print] doi:10.3109/09553002.2013.765055 [12] Goessler UR, Bugert P, Kassner S, Stern-Straeter J, Bran G, Sadick H, et al. In vitro analysis of radiation-induced dermal wounds. Otolaryngol Head Neck Surg 142:845-850; 2010. [13] Kim VN. MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6:376-385; 2005. [14] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281-297; 2004. [15] Cai Y, Yu X, Hu S, Yu J. A brief review on the mechanisms of miRNA regulation. Genomics Proteomics Bioinformatics 7:147-154; 2009. [16] Bushati N, Cohen SM. microRNA functions. Annu Rev Cell Dev Biol 2007;23:175-205. [17] Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet 10:704-714; 2009. [18] Zhang S, Song C, Zhou J, Xie L, Meng X, Liu P, et al. Amelioration of 17

radiation-induced skin injury by adenovirus-mediated heme oxygenase-1 (HO-1) overexpression in rats. Radiat Oncol 7:4; 2012. [19] Kohl RR, Kolozsvary A, Brown SL, Zhu G, Kim JH. Differential radiation effect in tumor and normal tissue after treatment with ramipril, an angiotensin-converting enzyme inhibitor. Radiat Res 168:440-445; 2007. [20] Rossi L, Reverberi D, Podesta G, Lastraioli S, Corvo R. Co-culture with human fibroblasts increases the radiosensitivity of MCF-7 mammary carcinoma cells in collagen gels. Int J Cancer 85:667-673; 2000. [21] Josson S, Sharp S, Sung SY, Johnstone PA, Aneja R, Wang R, et al. Tumor-Stromal Interactions Influence Radiation Sensitivity in Epithelial- versus Mesenchymal-Like Prostate Cancer Cells. J Oncol 2010;2010. pii: 232831. doi: 10.1155/2010/232831. Epub 2010 Jul 29. [22] Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, et al. The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1366:177-196; 1998. [23] Yang J, Wu LJ, Tashino S, Onodera S, Ikejima T. Reactive oxygen species and nitric oxide regulate mitochondria-dependent apoptosis and autophagy in evodiamine-treated human cervix carcinoma HeLa cells. Free Radic Res42:492-504; 2008. [24] Montero AJ, Jassem J. Cellular redox pathways as a therapeutic target in the treatment of cancer. Drugs 71:1385-1396; 2011. [25] Talapatra S, Wagner JD, Thompson CB. Elongation factor-1 alpha is a selective regulator of growth factor withdrawal and ER stress-induced apoptosis. Cell Death Differ 9:856-861; 2002. [26] Lenz H, Schmidt M, Welge V, Schlattner U, Wallimann T, Elsasser HP, et al. The creatine kinase system in human skin: protective effects of creatine against oxidative and UV damage in vitro and in vivo. J Invest Dermatol 124:443-452; 2005. [27] Tanaka S, Uehara T, Nomura Y. Up-regulation of protein-disulfide isomerase in response to hypoxia/brain ischemia and its protective effect against apoptotic cell death. J Biol Chem 275:10388-10393;2000. [28] Liu C, Li B, Cheng Y, Lin J, Hao J, Zhang S, et al. MiR-21 plays an important role in 18

radiation induced carcinogenesis in BALB/c mice by directly targeting the tumor suppressor gene Big-h3. Int J Biol Sci 7:347-363; 2011. [29] Shi Y, Zhang X, Tang X, Wang P, Wang H, Wang Y. MiR-21 is continually elevated long-term in the brain after exposure to ionizing radiation. Radiat Res 177:124-128; 2012. [30] Mert U, Ozgur E, Tiryakioglu D, Dalay N, Gezer U. Induction of p53-inducible microRNA miR-34 by gamma radiation and bleomycin are different. Front Genet 3:220;2012. [31] Liu C, Zhou C, Gao F, Cai S, Zhang C, Zhao L, et al. MiR-34a in age and tissue related radio-sensitivity and serum miR-34a as a novel indicator of radiation injury. Int J Biol Sci 7:221-233; 2011. [32] Irshad M, Chaudhuri PS. Oxidant-antioxidant system: role and significance in human body. Indian J Exp Biol 40:1233-1239; 2002. [33] Pugliese PT. The skin's antioxidant systems. Dermatol Nurs 10:401-416; quiz 417-408; 1998. [34] Jang J, Ye BR, Heo SJ, Oh C, Kang DH, Kim JH, et al. Photo-oxidative stress by ultraviolet-B radiation and antioxidative defense of eckstolonol in human keratinocytes. Environ Toxicol Pharmacol 34:926-934; 2012. [35] Robbins ME, Zhao W. Chronic oxidative stress and radiation-induced late normal tissue injury: a review. Int J Radiat Biol 80:251-259; 2004. [36] Schnackenberg CG. Oxygen radicals in cardiovascular-renal disease. Curr Opin Pharmacol 2:121-125; 2002. [37] Frank S, Munz B, Werner S. The human homologue of a bovine non-selenium glutathione peroxidase is a novel keratinocyte growth factor-regulated gene. Oncogene 14:915-921; 1997. [38] Munz B, Frank S, Hubner G, Olsen E, Werner S. A novel type of glutathione peroxidase: expression and regulation during wound repair. Biochem J 326 (Pt 2):579-585; 1997. [39] Kumin A, Huber C, Rulicke T, Wolf E, Werner S. Peroxiredoxin 6 is a potent cytoprotective enzyme in the epidermis. Am J Pathol 169:1194-1205; 2006. [40] Ewing D, Jones SR. Superoxide removal and radiation protection in bacteria. Arch 19

Biochem Biophys 254:53-62; 1987. [41] Kohen R, Gati I. Skin low molecular weight antioxidants and their role in aging and in oxidative stress. Toxicology 148:149-157; 2000. [42] Dayal D, Martin SM, Limoli CL, Spitz DR. Hydrogen peroxide mediates the radiation-induced mutator phenotype in mammalian cells. Biochem J 413:185-191; 2008. [43] Yan S, Brown SL, Kolozsvary A, Freytag SO, Lu M, Kim JH. Mitigation of radiation-induced skin injury by AAV2-mediated MnSOD gene therapy. J Gene Med 10:1012-1018; 2008. [44] Kanai AJ, Zeidel ML, Lavelle JP, Greenberger JS, Birder LA, de Groat WC, et al. Manganese superoxide dismutase gene therapy protects against irradiation-induced cystitis. Am J Physiol Renal Physiol 283:F1304-1312; 2002. [45] Doctrow SR, Lopez A, Schock AM, Duncan NE, Jourdan MM, Olasz EB, et al. A synthetic superoxide dismutase/catalase mimetic EUK-207 mitigates radiation dermatitis and promotes wound healing in irradiated rat skin. J Invest Dermatol 133:1088-1096; 2013. [46] Gu Q, Feng T, Cao H, Tang Y, Ge X, Luo J, et al. HIV-TAT mediated protein transduction of Cu/Zn-superoxide dismutase-1 (SOD1) protects skin cells from ionizing radiation. Radiation Oncology 8:253;2013.

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Figure legends Fig. 1. The 2-DE maps of rat skin tissues separated by pH 3-10 IPG strips. (A) and (B) Separation of proteins in normal and irradiated rat skin tissues. (C) Downregulation of PRDX-6 (indicated by white arrows) in 45 Gy irradiated skin tissues of rats. (D)-(I) Expression levels of select mRNAs were independently determined using real-time PCR in six pairs of skin specimens. Real-time PCR data were normalized to -Actin. I

(irradiated)/N (nonirradiated) relative mRNA expression ratios between normalized values were calculated. I/N values lower than 1 were transformed to the inverse value N/I. The + and - indicate that the expression is higher or lower, respectively, in the irradiated tissue sample than in the adjacent control skin tissue sample.

Fig. 2. MiRNA expression profiling of skin samples subjected to electron beam irradiation. (A) Heatmap of miRNA profiles showing an increase or decrease in expression following 45 Gy electron beam irradiation. The miRNAs marked with a red arrow were verified using quantitative assays. Annotations of the miRNAs are presented in Table 2. (B)-(F) Expression levels of select miRNAs identified by the microarray assay were independently determined using real-time PCR in an additional six pairs of skin specimens. Real-time PCR data were normalized to U6. I (irradiated)/N (nonirradiated)

relative miRNA expression ratios between normalized values were calculated. I/N values lower than 1 were transformed to the inverse value N/I. The + and - indicate that the expression is higher or lower, respectively, in the irradiated tissue sample than in the adjacent control skin tissue sample.

Fig. 3. PRDX-6 is a direct target of miR-214. (A) Putative miR-214-binding sequence in the 3’UTR of PRDX-6 mRNA. (B) and (C) 3’UTR region of PRDX-6 was cloned downstream of the luciferase reporter gene (pGL3-promoter). HaCaT cells (B) or primary rat skin cells (C) were cotransfected with the Firefly luciferase expression construct pGL3-PRDX6-UTR and either miR-214 mimics or a scrambled miRNA (miRNA-NC). HaCaT cells (B) or primary rat skin cells (C) were then cotransfected with 21

pGL3-PRDX6-UTR and either the miR-214 inhibitor or the control miRNA inhibitor NC. Luciferase activity was assayed 24 h after transfection. The Firefly luciferase activity of each sample was normalized by Renilla luciferase activity. The normalized luciferase activity of the cells transfected with scrambled miRNA was set as 100% relative luciferase activity. The column graphs show the means of at least three independent experiments performed in duplicate (* P < 0.05 and ** P < 0.001, compared with cells transfected with control miRNA). (D) Primary rat skin cells were transfected with the indicated RNAs for 24 h. Endogenous PRDX-6 and internal standard GAPDH protein levels were detected by western blotting using anti-PRDX-6 or anti-GAPDH primary antibodies.

Fig. 4. PRDX-6 modulates radiosensitivity, mitochondrial integrity and apoptosis of HaCaT cells. (A) HaCaT cells were transfected with the indicated vectors for 24 h and then were exposed to 2, 4, 6 or 8 Gy irradiation. The survival data were normalized to those of the nonirradiated control group. (B) Determination of ROS levels of HaCaT cells transfected with the indicated vectors. Fluorescent signals, reflecting the concentration of ROS, were measured by a fluorescence microscope under the same conditions. (C) Relative ROS level in indicated group of cells as calculated by ImageJ image analysis software (MD, USA). (D) Determination of mitochondrial integrity of HaCaT cells transfected with the indicated vectors. (E) Relative fluorescence intensity in indicated groups. (F) The effect of PRDX-6 on apoptosis in HaCaT cells. Cells were transfected with pcDNA-PRDX6 or control vector for 24 h and then exposed to 20 Gy of irradiation. Statistical analyses between the groups were determined by ANOVA (* P < 0.05, ** P < 0.01).

Fig. 5. miR-214 Inhibition decreases ROS and mitochondrial damage of HaCaT cells. HaCaT cells were transfected with the miRNA inhibitor negative control (NC) or miR-214 inhibitor for 24 h and then were exposed to 20 Gy irradiation. (A) Determination of ROS levels of HaCaT cells transfected with the indicated RNAs. Fluorescent signals, reflecting the concentration of ROS, were measured by a fluorescence microscope under the same 22

conditions. (B) Relative ROS level in indicated group of cells as calculated by ImageJ image analysis software. (C) Determination of mitochondrial integrity of HaCaT cells transfected with the indicated RNAs. (D) Relative fluorescence intensity in indicated groups. Statistical analyses between the groups were determined by ANOVA (* P < 0.05, , ** P < 0.01).

Fig. 6. PRDX-6 overexpression attenuates ROS and MDA after irradiation. Rat buttock skin was left without irradiation or irradiated with a single dose of 45 Gy electron beam irradiation followed by in vivo transfection with pcDNA3.1 or pcDNA3.1-PRDX-6 (6 animals per group). (A) Relative ROS levels in rat skin. Twenty-four hours after irradiation, the skin ROS levels were determined as described in the Materials and Methods. (B) Twenty-four hours after irradiation, the MDA concentration levels in the rat skin of different groups. The * denotes P < 0.05 compared with the PBS-injected control group.

Fig. 7. PRDX-6 overexpression ameliorates radiation-induced skin injury. Rat buttock skin was irradiated with a single dose of 45 Gy electron beam irradiation followed by in vivo transfection with pcDNA3.1 or pcDNA3.1-PRDX-6 (6 animals per group). (A) Skin injury in these groups was measured using a semi-quantitative score of 1 (no damage) to 5 (severe damage). (B) Representative skin images of different groups 30 days after irradiation. (C) Representative skin images of different groups 30 days after irradiation. (D) Relative epidermis thickness of rat skins 60 days after irradiation. (E) Representative H&E staining of rat skins 60 days after irradiation.

Table 1 Differentially expressed proteins in irradiated skin tissues of rats Table 2 Summary of significantly differentially expressed miRNAs in irradiated skin tissues compared to adjacent nonirradiated tissues

Supplementary Table 1 Primer sequences for real-time PCR analysis Supplementary Table 2 Summary of differentially expressed miRNAs in irradiated 23

skin tissues compared to adjacent nonirradiated tissues

24

3.1227 1.8733 1.7100 0.1301 0.1457 0.2169 0.2187 0.2296 0.2766 0.2815 0.2865 0.2881 0.2898 0.2905 0.2997 0.3209 0.3308 0.3456 0.3459 0.3532 0.3571 0.3634 0.3815 0.3899

Upregulated Upregulated Upregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated

Fold Changea

25

mitochondrial aspartate aminotransferase hemopexin precursor ATP synthase  subunit protein disulfide isomerase elongation factor-1 peptidyl-prolyl cis-trans isomerase A heat shock protein 1 heme-binding protein 23 Similar to 14-3-3 protein  serum albumin precursor calcium-binding protein 1 pyruvate kinase isozymes M1/M2 transitional endoplasmic reticulum ATPase creatine kinase, partial creatine kinase M-type purine nucleoside phosphorylase T-complex protein 1 subunit epsilon eukaryotic translation initiation factor 5A-1 isoform B collagen 1 78 kDa glucose-regulated protein precursor peroxiredoxin-6 (PRDX-6) rCG55067 dnaK-type molecular chaperone hsp72-ps1 transferrin

Protein name

irradiated skin tissues vs adjacent nonirradiated skin tissues

a

504 5715 6320 8120 603 1120 3206 2211 7123 5603 3510 2726 5401 1119 2812 3307 5707 6106 2923 5601 4205 2805 5727 2734

Spot ID

PI 3.4321 7.3619 8.0548 9.0184 3.1247 4.2804 6.1835 4.8761 8.8783 7.2670 5.6411 4.8246 7.1847 4.2333 4.7444 5.4151 7.2293 7.9690 4.8773 7.2223 6.7953 4.6194 7.5326 4.9351

Table 1 Differentially expressed proteins in irradiated skin tissues of rats 41.0453 73.8540 28.9313 18.2198 48.9422 15.2952 23.5574 22.8093 19.9242 51.5470 42.5542 59.7906 35.5111 18.1414 137.4085 30.9607 58.5700 16.8773 177.0707 53.8203 25.1464 104.7192 62.6956 75.9776

Mr (KD)

Table 2 Summary of significantly differentially expressed miRNAs in irradiated skin tissues compared to adjacent nonirradiated tissues Upregulated miRNAs miRNA ID

Fold Changea

P-value

rno-miR-223 rno-miR-34a rno-miR-142-3p rno-miR-196a rno-miR-21 rno-miR-214 rno-miR-199a-3p rno-miR-30d

5.0203 4.7964 4.7420 4.6848 3.8288 2.0316 1.8388 1.4403

0.0197 0.0035 0.0089 0.0150 0.0037 0.0383 0.0385 0.0030

Downregulated miRNAs miRNA ID

rno-miR-17-5p rno-miR-20a rno-miR-301a rno-miR-872* a

Fold Changea

P-value

0.1037 0.3685 0.3457 0.7104

0.0249 0.0110 0.0435 0.0166

irradiated skin tissues vs adjacent nonirradiated skin tissues

26

Figure 1

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Figure 4

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Figure 6

Figure 7