Differential Expression of Stress and Immune Response Pathway

Published OnlineFirst April 30, 2014; DOI: 10.1158/1541-7786.MCR-13-0623

Molecular Cancer Chromatin, Gene, and RNA Regulation Research

Differential Expression of Stress and Immune Response Pathway Transcripts and miRNAs in Normal Human Endothelial Cells Subjected to Fractionated or Single-Dose Radiation

Sanjeewani T. Palayoor1, Molykutty John-Aryankalayil1, Adeola Y. Makinde1, Michael T. Falduto2, Scott R. Magnuson2, and C. Norman Coleman1

Abstract Although modern radiotherapy technologies can precisely deliver higher doses of radiation to tumors, thus, reducing overall radiation exposure to normal tissues, moderate dose, and normal tissue toxicity still remains a significant limitation. The present study profiled the global effects on transcript and miR expression in human coronary artery endothelial cells using single-dose irradiation (SD, 10 Gy) or multifractionated irradiation (MF, 2Gy 5) regimens. Longitudinal time points were collected after an SD or final dose of MF irradiation for analysis using Agilent Human Gene Expression and miRNA microarray platforms. Compared with SD, the exposure to MF resulted in robust transcript and miR expression changes in terms of the number and magnitude. For data analysis, statistically significant mRNAs (2-fold) and miRs (1.5-fold) were processed by Ingenuity Pathway Analysis to uncover miRs associated with target transcripts from several cellular pathways after irradiation. Interestingly, MF radiation induced a cohort of mRNAs and miRs that coordinate the induction of immune response pathway under tight regulation. In addition, mRNAs and miRs associated with DNA replication, recombination and repair, apoptosis, cardiovascular events, and angiogenesis were revealed.

Implications: Radiation-induced alterations in stress and immune response genes in endothelial cells contribute to changes in normal tissue and tumor microenvironment, and affect the outcome of radiotherapy. MolCancerRes;12(7);1002–15. 2014 AACR.

Introduction to tumor focusing on areas deemed at highest risk (3–5). The Radiation oncology remains a mainstay of cancer therapy newer technology can reduce high doses to normal tissues as both curative and palliative therapy used alone or as a but can increase the amount of tissue receiving daily dose (6). component of combined modality therapy. Routinely, in The incidental radiation exposure of normal tissues is a topic clinical practice, radiation therapy is administered as mul- of concern in radiotherapy (7). tiple fractions of 2 to 2.5 Gy per day for 5 days per week for 1 Recent work from our laboratory showed that prostate to 7 weeks to allow repair, repopulation, and recovery of the carcinoma cells that survive multifractionated (MF) radiation exposure have a different genomic signature compared collateral damage to the normal tissue (1, 2). Individualized – radiation therapy with development of modern techniques with the cells exposed to single-dose radiation (SD; refs. 8 10). Exposure to 10 Gy radiation delivered as fractionated such as intensity-modulated radiation therapy and image- guided radiation therapy can deliver more controlled single irradiation (1 Gy 10 or 2 Gy 5) resulted in more robust or fewer fractions of high-dose radiation (hypofractionation) differential gene expression changes in PC3 and DU145 cells, whereas in LNCaP cells, 10 Gy radiation delivered as a single dose was more effective (9, 10). These studies also Authors' Affiliations: 1Radiation Oncology Branch, Center for Cancer revealed that the mRNA expression profiles following frac- Research, National Cancer Institute, NIH, Bethesda, Maryland; and 2GenUs fl Biosystems, Inc., Northbrook, Illinois tionated irradiation were in uenced by p53 status. In LNCaP cells, harboring wild-type p53 DNA replication/ Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). recombination/repair and cell cycle were the top gene ontology categories affected by radiation, whereas in p53- Corresponding Author: Sanjeewani T. Palayoor, National Cancer Insti- tute, NIH, 9000 Rockville Pike, Building # 10, Room B3B406, Bethesda, MD mutated PC3 cells, genes from IFN, immune, and stress 20892. Phone: 301-496-1401; Fax: 301-480-1434; E-mail: response categories were altered significantly. miRNAs play [email protected] an important role in regulation of gene expression at the doi: 10.1158/1541-7786.MCR-13-0623 posttranscriptional level by base pairing with the comple- 0 2014 American Association for Cancer Research. mentary sequences within 3 -untranslated regions of target

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mRNAs, resulting in translational repression or mRNA SD and 6 and 24 hours after the final dose of fractionated degradation (11). As observed for the mRNA expression irradiation. Separate controls were maintained for SD and profiles, in the prostate carcinoma cells, treatment with MF radiation protocols. fractionated irradiation significantly altered more miRNAs as compared with the cells exposed to SD radiation (12). RNA isolation Although normal tissue exposure remains a major con- Cells were pelleted at 6 and 24 hours after an SD and 6 fi cern in radiation therapy, few studies have investigated the and24hoursafterthe nal dose of MF irradiation and molecular effects of various radiation treatment regimens stored in liquid nitrogen. Total RNA including small in normal cells. The purpose of the present study was to RNAs was isolated using phenol/chloroform extraction fi investigate global gene and miRNA alterations in normal followed by puri cation over spin columns (Ambion Cat. cells exposed to radiation protocols simulating hypofrac- No. AM9738). The concentration and purity of total tionated and conventionally fractionated radiation regi- RNA were measured by spectrophotometry at OD260/ mens typically used for radiotherapy in clinic. For this 280, and the quality of the total RNA sample was assessed study, we treated normal human coronary artery endo- using an Agilent Bioanalyzer with the RNA6000 Nano thelial cells (HCAEC) with 10 Gy radiation delivered as a Lab Chip (Agilent Technologies). SDradiationoras5fractionsof2Gyradiation(MF).The mRNA microarray analysis differentially expressed mRNAs and miRNAs were iden- The mRNA microarray analysis was performed using tified by microarray analysis at 6 and 24 hours after an SD Agilent Technologies Human Gene Expression 4 44 K and 6 and 24 hours after the final dose of fractionated V2 microarrays (product number G4845A, design irradiation. These data showed that in HCAEC more ID 026652) designed to target 27,958 Entrez Gene RNAs. mRNAs and miRNAs were differentially expressed by exposure to MF compared with SD, and the magnitude miRNA microarray analysis of changes was higher in MF-irradiated cells. Gene ontol- The miRNA microarray analysis was performed using ogy classification showed that in addition to cell cycle, Agilent Technologies Human miRNA 8 15 K V2 micro- genes regulating DNA replication, DNA damage stimu- arrays (product number G4470B, design ID 019118) with lus, and DNA repair, and genes related to immune probes for 723 human and 76 human viral miRNAs sourced response were significantly altered following exposure to from Sanger miRBase (release 10.1). MF. Using ingenuity target filter program, we identified The mRNA and miRNA microarray data were analyzed miRNAs associated with the target genes from different using Gene Spring Software (Agilent Technologies) as cellular pathways that were differentially expressed in described previously (12). To ensure that mRNAs and response to SD and MF. The present study suggests that miRNAs were reliably measured, ANOVA was used to endothelial cells may play an important role in the out- compare the means of each condition (n ¼ 3). For mRNA come of radiotherapy in the clinical settings. analysis, cutoff ratios of gene expression greater than 2.0 and less than 0.5 and a P value of <0.05 relative to the respective Materials and Methods control group were selected. For miRNA analysis, cutoff Cells ratios greater than 1.5 and less than 0.66 with a P value of Cryopreserved HCAEC and the media were purchased <0.05 relative to the respective control were selected. from Lonza Walkersville Inc. Cells were thawed and main- The mRNA and miRNA microarray data discussed in this tained in EBM-2 basal medium supplemented with FBS and publication have been deposited in NCBI's Gene Expression growth factors (EGM-2 MV BulletKit CC-3202) according Omnibus and are accessible through GEO Series Accession to the supplier's instructions. Cells from passages P1 to P3 No. GSE57059 (http://www.ncbi.nlm.nih.gov/geo/query/ were used. acc.cgi?acc¼GSE57059), and Accession No. GSE56824 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? Radiation acc¼GSE56824), respectively. Cells were irradiated in a PANTAK high-frequency X-ray generator (Precision X-ray Inc.), operated at 300 kV and Real-time RT-PCR 10 mA. The dose rate was 1.6 Gy per minute. Cells were Separate experiments were set up to extract RNA at 6 and plated into T75 cm2 flasks (1–1.5 106 for SD radiation 24 hours after an SD and 6 and 24 hours after the final dose and 0.6–0.8 106 for fractionated radiation). After 24 of fractionated irradiation for real-time RT-PCR analysis. hours, cells were exposed to a total of 10 Gy radiation RNA was isolated using the RNAeasy mini Kit (Cat. No administered either as a SD radiation or as MF radiation 74104; Qiagen) as described previously (12). Purified RNA of 2 Gy 5. These nonisoeffective doses were selected to was reverse transcribed to cDNA and RT-PCR was carried simulate clinical hypofractionated and conventionally frac- out as described previously (13). Alterations in selected tionated radiotherapy regimens. For the MF protocol, cells differentially expressed genes were confirmed using Taq- were exposed to 2 Gy radiation twice a day, at 6-hour Man Custom Express Plate (Part # 4391524; Applied interval. The cells were approximately 90% confluent at Biosystem) and ABI PRISM 7500 Sequence Detection the time of harvesting. For both protocols, radiation- System instrument equipped with the SDS version 1.4 induced changes were analyzed at 6 and 24 hours after an software. Each plate was designed to contain 18S and PES1

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endogenous controls and 22 individual Taqman Gene keeping with the selected SD regimen having a higher Expression Assays in quadruplets in specified well locations biologically effective dose than the MF regimen. (see Supplementary Data for assay IDs and expanded method). Gene expression analyses in HCAEC following SD and MF irradiation Ingenuity Pathway Analysis Global gene expression changes. Of the total 27,958 The functional significance of differentially expressed genes represented in the Agilent microarrays, treatment with mRNAs (2-fold change and P < 0.05) following SD and 10 Gy SD and 2 Gy 5 MF resulted in differential MF irradiation was evaluated using Ingenuity Pathway Anal- expression (>2 fold, P < 0.05) of combined 2,255 genes in ysis (IPA) software (Ingenuity Systems Version 8.7-3203) as HCAEC cells. The Venn diagrams and the heat map described previously (12, 13). Datasets were uploaded into in Fig. 1 show that the MF exposure resulted in more robust the IPA, which were next mapped to the functional networks gene expression changes compared with the SD radiation available in the Ingenuity Pathway Knowledge Base and (Fig. 1). Of the total 2,255 genes altered, 89 genes were ranked by score as described previously (12). differentially expressed in response to SD, 1,873 genes were differentially expressed in response to MF, and 293 genes miRNA target filter analysis were commonly differentially expressed following SD and To identify the target mRNAs associated with differen- MF treatment (Fig. 1A and B). In cells irradiated with SD, tially expressed miRNAs, datasets of differentially expressed more genes were differentially expressed at 24-hour com- miRNAs (1.5-fold change and P < 0.05) and differentially pared with 6-hour time point (Fig. 1C). Significant gene expressed mRNAs (2-fold change and P < 0.05) were expression changes were evident at 6 hours in cells irradiated uploaded into an IPA "MicroRNA Target Filter" program. with MF, and the changes persisted up to 24 hours (Fig. 1C). For the data analysis, only the experimentally verified and Enrichment of genes by gene ontology classification. highly predicted targets from IPA database were selected. The 2,255 differentially expressed genes were classified in to functional categories by gene ontology classification (Sup- Cell-cycle analysis plementary Table S1). The enrichment factor of the cell- Cells were fixed in 70% ethanol 6 and 24 hours after an cycle regulatory genes was the highest. The other signifi- SD and 6 and 24 hours after the final dose of MF. Data were cantly altered categories were stress response, DNA replica- collected and analyzed as described previously (13). tion, response to DNA damage, DNA repair, immune response, apoptosis, p53, and inflammatory response. The Western blotting number of genes altered and the magnitude of change in Cell extracts were prepared 6 and 24 hours after an SD and these categories were much higher after exposure to MF than 6 and 24 hours after the final dose of MF. Proteins were SD. Genes from cytokines, inflammatory response, and separated and protein bands were captured by digital CCD growth factor activity categories that could influence other camera (Fuji, LAS 3000) as described previously (13). The cells and tissues were significantly altered only by MF. membranes were stripped and reprobed for actin. Signal intensities were quantified using Image J 1.44p software (NIH), normalized to the loading control actin, and AC expressed as fold change compared with the unirradiated Up in SD Up in MF SD MF control. 6 h 24 h 6 h 24 h Antibodies 53 116 1,107 p53 (Sc-6243), p21 (Sc-756), MDM2 (Sc-5304), RAD51 (Sc-8349) and STAT-1 (Sc-346; Santa Cruz Biotechnology, Inc.), cyclin D2 (3741) and caspase 1 (3866; Cell Signaling Technology), and actin (MAB1501R; Millipore). B Down in SD Down in MF Data analysis Each data point represents average SEM of 3 experiments. Differences between the groups were statistically 36 177 766 evaluated by two-tailed paired t test. A P value of <0.05 was considered statistically significant. 0.25 1 4 mRNA (2,255; 2-fold, P < 0.05) Results Surviving fractions of HCAEC following SD and MF Figure 1. Venn diagrams (A and B) depict the numbers of differentially irradiation expressed genes (>2 fold change, P < 0.05) in HCAEC exposed to 10 Gy SD and 2 Gy 5 MF radiation. Genes upregulated (A) and downregulated The surviving fraction (SF) of HCAEC exposed to 2 Gy (B) by SD and MF irradiation. C, heat map of differentially expressed 5 MF irradiation was 0.003 0.0002 and for those exposed genes at 6 and 24 hours after an SD and 6 and 24 hours after the final dose to 10 Gy SD irradiation was 0.00008 0.00002. This is in of MF irradiation. Yellow to red, upregulated; blue, downregulated genes.

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Table 1. Functions associated with top 10 networks of genes differentially expressed in irradiated HCAEC

Radiation ID Score Top functions SD 6 h 1 66 Cellular development, hematopoiesis, cell death (27) 2 31 Cell cycle, cancer, cell death (16) 3 20 Cell cycle, molecular transport, protein synthesis (11) 4 20 Cellular function and maintenance, cell cycle, connective tissue development and function (11) SD 24 h 1 53 Cellular assembly and organization, DNA replication, recombination, and repair, cell cycle (29) 2 40 DNA replication, recombination, and repair, cell cycle, cancer (24) 3 34 Cell death, free radical scavenging, lipid metabolism (21) 4 33 Free radical scavenging, drug metabolism, endocrine system development and function (21) 5 30 Cell morphology, cancer, reproductive system disease (20) 6 30 Cellular movement, cell morphology, cell-to-cell signaling and interaction (20) 7 28 Cellular assembly and organization, cellular compromise, cell morphology (21) 8 25 Cellular development, cellular growth and proliferation, cell cycle (17) 9 24 Cellular compromise, cancer, hematologic disease (17) 10 23 Cellular development, hematopoiesis, cell death (18) MF 6 h 1 45 Cell cycle, cellular assembly and organization, DNA replication, recombination, and repair (34) 2 40 Cellular assembly and organization, DNA replication, recombination, and repair, amino acid metabolism (32) 3 38 Infectious disease, DNA replication, recombination, and repair, gene expression (31) 4 35 Cell cycle, genetic disorder, ophthalmic disease (30) 5 35 Cell cycle, cellular assembly and organization, cellular function and maintenance (30) 6 35 Small molecule biochemistry, lipid metabolism, molecular transport (30) 7 33 DNA replication, recombination, and repair, cell cycle, cellular assembly and organization (29) 8 32 Cellular assembly and organization, cellular function and maintenance, DNA replication, recombination, and repair (30) 9 31 Cellular assembly and organization, DNA replication, recombination, and repair, cell cycle (28) 10 31 Cell cycle, cell morphology, cellular function and maintenance (30) MF 24 h 1 50 Cellular growth and proliferation, cancer, gastrointestinal disease (34) 2 47 Cellular assembly and organization, DNA replication, recombination, and repair, cell cycle (33) 3 40 Cellular assembly and organization, DNA replication, recombination, and repair, cancer (30) 4 38 Cell cycle, cellular assembly and organization, DNA replication, recombination, and repair (29) 5 36 Infectious disease, dermatologic diseases and conditions, genetic disorder (28) 6 36 Cell cycle, cellular assembly and organization, DNA replication, recombination, and repair (28) 7 34 Cancer, genetic disorder, ophthalmic disease (27) 8 34 RNA posttranscriptional modiﬁcation, gene expression, genetic disorder (27) 9 30 DNA replication, recombination, and repair, cell cycle, cell death (27) 10 30 DNA replication, recombination, and repair, cell cycle, cellular development (25)

NOTE: Functions associated with networks of genes differentially expressed by SD and MF irradiation. IPA of differentially expressed genes in HCAEC treated with 10 Gy single (SD) and 2 Gy 5 fractionated (MF) irradiation at 6 h and 24 h after a SD and 6 and 24 h after the ﬁnal dose of MF irradiation. The network ID, score, number of focus molecules (in bracket) and the functions associated with top 10 networks with score >10 are shown.

IPA. Functions associated with top 10 networks (score > repair. RNA posttranscriptional modification was another 10) of genes differentially expressed by SD and MF are significant category observed at both time points following shown in Table 1. At 6 hours following SD, only 4 networks MF (Table 1). with score more than 10 were generated, with cell cycle and Cell-cycle analysis. Because cell cycle was the topmost cell death as the top functions. At 24-hour time point after category affected by radiation, cell-cycle distribution in the SD irradiation, there were 14 networks with scores more HCAEC treated with single and fractionated radiation was than 10. In addition to cell cycle, the other top functions examined. Figure 2 shows the distribution of cells in G1,S, included DNA replication, recombination, and repair. At 6- and G2 compartments after treatment with 10 Gy SD and 24-hour time point after the final dose of MF irradiation, (Fig. 2A) and 2 Gy 5 MF (Fig. 2B). Exposure to SD > > there were 20 networks with score 10. The main func- resulted in reduction in cells in G1 at 6 hours compared with tional category in cells treated with MF at 6- and 24-hour the untreated cells, which persisted at 24 hours. There was an time point included DNA replication, recombination, and increase in the percentage of cells in G2 at 24 hours. Exposure

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involved in homologous recombination from DNA repair A 70 category included H2AFX (H2AX), BRCA1, BRCA2, BARD1 RPA1 RAD51 RAD51AP RAD54B RAD54L 60 , , , , , , * and BLM. Other downregulated genes from DNA repair 50 * category were DNA polymerases POLA1, POLD2, POLD3, * POLE2,andPOLQ, DNA primase PRIM1, replication factor 40 RFC5, ribonucleotide reductases RRM1, RRM2,andDCK. RFWD3 30 The downregulated genes related to p53 included , % Cells GTSE1, BLM, BRCA1, BRCA2, HSPD1,andMTBP;the 20 upregulated genes related to p53 were PML, MDM2, C16orf5, CDKN1A (p21), ATM, TP53INP1,and 10 TP53INP2. 0 Figure 3B shows heat map of genes from immune response Control 10 Gy × 1 Control 10 Gy × 1 category, which includes inﬂammatory genes subset. In the 6 h 24 h immune response category, the majority of genes were upregulated following fractionated irradiation. These Time after radiation included adhesion molecules ICAM1 and VCAM1, chemo- CXCL10 CXCL11 CXCL12 CXCL16 CCL2 B 70 kines , , , , , CCL5, CCL20, and CCL23, cytokines IFNE, IFNA4, IL1A, 60 IL1B, IL15, TGFB1, and TGFB2, receptors for chemokines CXCR4 and CXCR7 and cytokines FAS, IFN-induced 50

40 * AB 30 % Cells Stress response Immune response

20 SD MF SD MF 6 h 24 h 6 h 24 h 6 h 24 h 6 h 24 h stimulus

10 Damage DNA (26)

0 Inflammatory Control 2 Gy × 5 Control 2 Gy × 5 6 h 24 h (45) Time after radiation DNA Repair G1 S G2 (86)

Figure 2. Cell-cycle perturbations in HCAEC exposed to SD (A; 10 Gy) and MF (B; 2 Gy 5) radiation at 6 and 24 hours after an SD and 6 and 24 hours after the ﬁnal dose of MF irradiation. , P < 0.05. Remaining immune response genes to MF resulted in reduction in the percentage of cells in S phase at 24 hours. Remaining stress response genes Heat maps. Radiation-induced changes in individual

genes from selected functional categories were color coded (107) to demonstrate the expression patterns of individual genes within a category for each radiation treatment (164) regimen. Figure 3A shows heat map of genes from stress response category, which includes DNA damage stimulus and DNA repair gene subsets and other stress response genes. The majority of genes from DNA damage stimulus and DNA repair subsets were downregulated in response to SD and MF. However, the magnitude of downregulation was 0.25 14 much higher with MF. Many of the downregulated genes following SD did not pass the cutoff (<2 fold, P > 0.05) because the ratio of fold change, although statistically sig- ﬁ Figure 3. Heat maps depicting differentially expressed genes from stress ni cant, was less than 2-fold. A complete list of genes from response (A; includes DNA damage stimulus, DNA repair, and other stress response category with fold changes is given in Sup- stress response genes) and immune response (B; includes inﬂammatory plementary Table S2. Some of the downregulated genes subset) categories following SD and MF irradiation in HCAEC.

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proteins and transcription factors, and molecules in integrin mRNA targets of the miRNAs differentially expressed in signaling pathways ITGA4, ITGB3, and ITGAV. Genes the irradiated HCAEC. IPA miRNA–mRNA target filter regulating HLA-A, B, C, F, G, and J MHC class I molecules program was used to identify the target mRNAs associated were upregulated following fractionated irradiation. From with the differentially expressed miRNAs in HCAEC treated HLA class II, HLA DPA1 and DPB1 were downregulated with SD and MF radiation. Only the highly predicted or and HLA DQB1 was upregulated. A complete list of genes experimentally verified targets were included for the target from immune response category with fold changes is given in analysis. The differentially expressed miRNAs and their Supplementary Table S3. target mRNAs showed inverse correlation (altered in oppo- site direction) as well as direct correlation (both altered in miRNA analyses in HCAEC following SD and MF same direction). irradiation Table 2A shows the differentially expressed miRNAs Global miRNA changes in HCAEC following SD and after an SD at 6 and 24 hours and the number of mRNA MF. The miRNA microarray analysis revealed that 123 targets of these miRNAs observed in the present data. At miRNAs were differentially expressed with high confidence 6-hour time point, 3 miRNAs were upregulated and 5 (>1.5 fold, P < 0.05) in the irradiated cells, and the majority mRNAs were downregulated showing inverse correlation of them were upregulated (Fig. 4A–C). Exposure to SD between the miRNAs and mRNAs. At the same time resulted in the differential expression of 17 miRNAs, where- point, 5 upregulated miRNAs showed direct correlation as exposure to MF altered 101 miRNAs. Five miRNAs were with 8 mRNAs that were also upregulated. At 24-hour commonly expressed after SD and MF irradiation. At 6-hour time point, 5 differentially expressed miRNAs showed time point, more miRNAs were differentially expressed in inverse correlation with 13 differentially expressed cells exposed to MF compared with the 24-hour time point, mRNAs. At the same time point, a total of 8 differentially and the majority of them were upregulated (Fig. 4C). These expressed miRNAs showed direct correlation with 16 included the members of tumor suppressor let-7 family (let- differentially expressed mRNAs. Exposure to MF altered 7a, let-7e, and let-7f). Tumor suppressor miR34a was com- more number of miRNAs and mRNAs compared with mon for SD and MF, and was upregulated at 24 hours after SD, especially at 6-hour time point (Table 2B). At 6 hours SD, and 6 and 24 hours after MF. The members of the after the finaldoseofMF,atotalof48miRNAswere oncomir miR17-92 cluster (miR17, miR18a, miR18b, differentially expressed (47 up and 1 down) and showed miR19a, miR19b, miR20a, and miR92a) were all down- inverse correlation with 617 target mRNAs (596 down regulated after MF at 24 hours. The miRNAs associated with and 21 up). Upregulated 44 miRNAs also showed direct cardiovascular functions (miR195, miR21, miR221, miR222, correlation with 997 upregulated mRNAs at this time miR27b, miR29b, all upregulated), hypoxia response point. At 24 hours after the final dose of MF treatment, 10 (miR210, miR424, upregulated), and senescence (downregu- miRNAs were upregulated and 10 were downregulated. lated: miR15a, miR20a; upregulated: miR410 and miR431) They showed inverse and direct correlation with 264 and were also differentially expressed in cells exposed to MF. 270 mRNAs, respectively. Pathway analyses. Table 3 shows the differentially expressed mRNAs from ATM, p53 signaling and cell cycle AC checkpoint pathways, and the inversely regulatory miRNAs Up in SD Up in MF SD MF associated with these mRNA targets, identified by IPA target fi 6 h 24 h 6 h 24 h lter analysis. Most of the genes from ATM signaling pathway were downregulated in cells treated with MF 16 4 78 (Table 3). Activation of H2AFX (H2AX) immediately after DNA double-strand break results in recruitment of specific DNA repair proteins in ATM signaling pathway. H2AX was downregulated at 6 hours after the final dose of MF (MF 6 B h), and its regulatory miRNA miR24 was upregulated. Down in SD Down in MF Several other genes, including CDC25A, FANCD2, SMC1A, SMC2, BRCA1, CHEK1, CDK1, and CCNB1, were downregulated after MF exposure. The miRNAs show- 1123 ing inverse correlation with these target genes are shown in the table (Table 3). CDC25A was downregulated after both miRNA 0.25 1 4 (123; 1.5-fold, P < 0.05) SD and MF, and its regulatory miRNA miR34a was upregulated in SD 24 h, MF 6 h, and MF 24 h. However, at MF 6 h in addition to miR34a several other miRNAs regulating Figure 4. Venn diagrams (A and B) depict the numbers of differentially CDC25A were also upregulated. At 6 hours after MF, expressed miRNAs (>1.5 fold change, P < 0.05) in HCAEC exposed to 10 BRCA1 showed inverse correlation with miR146a as well Gy SD and 2 Gy 5 MF radiation. A, miRNAs upregulated by SD and MF. B, miRNAs downregulated by SD and MF irradiation. C, heat map of as miR24, but only with miR146a at 24-hour time point. In differentially expressed miRNAs at 6 and 24 hours after an SD and 6 and addition to BRCA1, miR24 inversely correlated also with 24 hours after the final dose of MF irradiation. E2F2 and CDK1. miR17-92 cluster was downregulated at

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Table 2. Number of mRNA targets showing inverse and direct correlations with differentially expressed miRNAs at 6 h and 24 h following

A. SD irradiation

SD 6 h SD 24 h

Inverse correlation Direct correlation Inverse correlation Direct correlation

miRNA Targets miRNA Targets miRNA Targets miRNA Targets miR326 1 miR329 1 miR1225-5p 1 miR1225-5p 1 miR329 1 miR484 1 miR136 2 miR136 1 miR32 3 miR532-5p 2 miR326 2 miR140-3p 1 3 up 5 down miR543 2 miR34a 3 miR326 5 miR32 2 miR7 5 miR338-3p 3 5 up 8 up 4 up 8 down miR34a 2 1 down 5 up miR874 1 miR7 2 7up 14up 1 down 2 down B. MF irradiation

MF 6 h MF 24 h

Inverse correlation Direct correlation Inverse correlation Direct correlation

miRNA Targets miRNA Targets miRNA Targets miRNA Targets

let-7a/f 28 let-7a/f 54 miR137 22 miR137 25 miR101 10 miR101 41 miR140-3p 2 miR181a 26 miR103 12 miR103 21 miR146a 19 miR140-3p 4 miR136 12 miR136 17 miR154 7 miR146a 13 miR137 20 miR137 41 miR181a 17 miR154 5 miR140-5p 6 miR140-5p 14 miR23a/b 37 miR23a/b 28 miR146a 15 miR146a 26 miR31 12 miR31 9 miR195 41 miR195 41 miR338-3p 6 miR338-3p 14 miR181a 21 miR181a 51 miR34a 11 miR34a 13 miR185 7 miR185 25 miR1275 12 miR1275 4 miR193a-5p 4 miR193a-5p 3 miR16 28 miR16 31 miR21 12 miR21 22 miR18a/b 11 miR18a/b 9 miR22 10 miR210 1 miR19a/b 27 miR19a/b 30 miR222/221 8 miR22 14 miR17/20a 28 miR17/20a 33 miR23a/b 40 miR222/221 17 miR7 9 miR7 12 miR24 14 miR23a/b 51 miR92a 16 miR92a 14 miR26a/b 20 miR24 19 miR27b/a 30 miR26a/b 38 10 up 133 down 10 up 137 up miR299-5p 3 miR27b/a 54 10 down 131 up 10 down 133 down miR29b 18 miR299-5p 3 miR30b 27 miR29b 49 miR326 11 miR30b 58 miR329 10 miR326 23 miR338-3p 8 miR329 21 miR342-3p 8 miR338-3p 18 miR410 32 miR342-3p 9 miR361-5p 8 miR410 41 (Continued on the following page)

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Table 2. Number of mRNA targets showing inverse and direct correlations with differentially expressed miRNAs at 6 h and 24 h following (Cont'd)

B. MF irradiation

MF 6 h MF 24 h

Inverse correlation Direct correlation Inverse correlation Direct correlation

miRNA Targets miRNA Targets miRNA Targets miRNA Targets

miR365 8 miR361-5p 14 miR374a/b 31 miR365 14 miR376c 5 miR374a/b 46 miR377 18 miR376c 26 miR379 1 miR377 27 miR409-3p 7 miR379 6 miR409-5p 2 miR409-3p 12 miR431 2 miR431 10 miR34a 11 miR34a 30 miR487b 2 miR487b 2 miR495 42 miR495 38 miR543 25 44 up 997 up miR654-3p 4 miR28-5p 3 miR7 21 47 up 596 down 1 down 21 up

NOTE: Differentially expressed miRNAs and the number of mRNA targets showing inverse and direct correlations with each miRNA. The differentially expressed mRNA targets of the differentially expressed miRNAs were identiﬁed using IPA miRNA/mRNA target ﬁlter analysis program. The table gives the number of mRNAs showing inverse and direct correlations with the miRNA differentially expressed at 6 and 24 h after 10 Gy single-dose (SD; A) and 2 Gy 5 fractionated (MF; B) irradiation. Upregulated miRNAs and mRNAs are shown in bold.

24 hours after MF irradiation. At this time point, p53- showed inverse correlation with CCND2. CCNE2 was regulated targets CCND2, CDKN1A (p21), and SERPINE2 downregulated and showed inverse correlation with miR34. were upregulated showing inverse correlation with members Several other miRNAs (miR30b, miR374a/b, and miR495) of the miR17-92 cluster and miR16. Other upregulated gene also showed inverse correlation with CCNE2 at 6 hour after in p53 pathway was FAS, and it showed inverse correlation MF. miR195 inversely correlated with targets CDC25B, with miR1275. miR23a/b was upregulated and showed CHEK1, and PLK1. Other miRNAs and their inverse targets inverse correlation with TOPBP1. associated with cell-cycle checkpoints were PKMYT1/ The mRNA targets associated with cell-cycle checkpoints miR27b/a, TOP2A/miR410, CKS1B/miR361-5p, and and miRNAs inversely correlated with these targets are CCNB1/miR379. shown in Table 3. E2F2 was commonly downregulated in Table 3 also demonstrates that at some time points, all radiation treatment groups and showed inverse correla- although target genes were differentially expressed, no tion with miR326 after SD 6 h, SD 24 h, and MF 6 h. In regulatory miRNA were identiﬁed. For example, PLK1 addition to miR326, several other miRNAs (let-7a/f, miR24, was downregulated in SD 6, SD 24, MF 6, and MF 24. miR222/221, miR365, and miR495) also showed inverse However, miR195, which showed inverse correlation correlation with E2F2 in MF at 6 hours. However, 24 hours with PLK1, was differentially expressed only in MF at after fractionated irradiation these miRNAs were no longer 6 hours. differentially expressed, and E2F2 showed inverse correla- Immune response pathway. Table 4 shows the differ- tion with miR31. CCND2 was upregulated at 6 and 24 entially expressed genes from immune response category that hours in cells treated with fractionated radiation. However, showed inverse correlations with the differentially expressed at 6-hour time point, all the miRNAs associated with miRNAs in the present microarray data by target ﬁlter CCND2 were also upregulated (not shown). At 24-hour analysis. At 24 hours following SD, miR7 showed inverse point, several miRNAs from miR17-92 cluster and miR16 correlation with RELB. Some of the immune response genes

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Table 3. Target ﬁlter analysis of target genes and miRNAs from ATM and P53 signaling pathways and cell cycle check points showing inverse correlation after single and fractionated irradiation

Pathways and mRNA SD 6 h SD 24 h MF 6 h MF 24h check points Target miRNA miRNA miRNA miRNA ATM H2AFX# — NC miR24" —

ATM, G1–S CDC25A# — miR34a" miR365", miR34a", let-7a/f", miR195" miR34a" ATM FANCD2# ——miR21", let-7a/f", miR23a/b" miR23b" ATM SMC1A# ——let-7a/f", miR137", miR342-3p" miR137" ATM SMC2# ——miR410" NC ATM, P53, BRCA1# ——miR146a", miR24" miR146a" G2M ATM, P53, CHEK1# ——miR195" NC G2M ATM, G2M CCNB1# NC — miR379" NC ATM, P53, CDKN1A" NC — NC miR20#, miR17# G1S

P53, G1–S E2F1# ——miR136", miR21" NC P53 TOPBP1# NC NC miR23a/b" miR23b"

P53, G1–S CCND2" NC NC NC miR16#, miR17#, miR18a/b#, miR19b/a#, miR20a/b# P53 SERPINE2" ——NC miR16# P53 FAS" NC NC NC miR1275#

G1–S E2F2# miR326" miR326" let-7a/f", miR24", miR326", miR31" miR222/221", miR365", miR495"

G1–S CCNE2# — miR34a" miR34a", miR30b",miR374a/b", miR34a" miR495" G2M CDC25B# ——miR195" NC G2M PLK1# NC NC miR195" NC ATM, G2M CDK1# ——miR24", miR410" NC G2M PKMYT1# ——miR27a/b" NC G2M TOP2A# ——miR410" NC G2M CKS1B# ——miR361" NC

NOTE: Target ﬁlter analysis of target genes and regulatory miRNAs showing inverse correlations following single and fractionated irradiation. Target mRNA and regulatory miRNA from ATM, P53 signaling and Cell cycle check point pathways. Upregulated target genes and miRNAs are shown in bold. Abbreviation: NC, only the target mRNA was differentially expressed, no corresponding differentially expressed miRNA identiﬁed. —, Neither the target mRNA nor any corresponding miRNA were differentially expressed in that protocol.

and the inversely correlated miRNAs in cells exposed diseases and angiogenesis in HCAEC are given in the to fractionated radiation were chemokines CXCL10/miR16, Supplementary Data. MiRNAs and genes associated with CXCL12/miR19b/a, cytokine TNFSF9/miR16 and cyto- cardiac hypertrophy, hypoxia signaling, atherosclerosis kine receptors TNFRSF9/miR1275, TNFRSF1B/miR338- signaling, and b adrenergic signaling in cardiovascular 3p, and genes associated with integrin signaling ITGB3/ pathway are shown in Supplementary Table S4. Many miR19b/a, ITGA4/miR20a/miR17-5p, and ITGAV/ of the miRNAs differentially expressed in HCAEC treated miR92a. Expression of IFN regulatory transcription factor with fractionated irradiation have been implicated in IRF9 was inversely correlated with miR20. Other prominent cardiovascular events and angiogenesis and are shown in upregulated immune response genes and the miRNAs Supplementary Table S5. inversely correlating with them were FAS/miR1275, Conformation of mRNA microarray data. Selected STAT2/miR19b/a, and PTGS2/miR16. IL1RAP, associated differentially expressed stress and immune genes from the with synthesis of acute phase and proinﬂammatory proteins, microarray data were analyzed by real-time RT-PCR. The was downregulated and showed inverse correlation with RT-PCR data substantially conﬁrmed the microarray data upregulated miR31 and miR146a. (Supplementary Table S6). Cardiovascular pathway. Radiation-induced differ- The expression of selected differentially expressed genes, entially expressed miRNAs associated with cardiovascular P53, CDKN1A, MDM2, RAD51, Cyclin D2, CASP1, and

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mRNA and miRNA Proﬁles of Irradiated Endothelial Cells

Table 4. Target ﬁlter analysis of target genes and miRNAs from immune response pathway showing inverse correlation after single and fractionated irradiation

SD 24 h MF 6 h MF 24 h

Targets miRNA Targets miRNA Targets miRNA CCNE2# miR34a" CCNA2# miR146a", miR24", miR410" COL1A2" miR7#, miR92a# RELB" miR7# CCNE2# miR30b", miR374a", miR34a", CCNA2# miR146a" miR495" COL1A2" miR7# CCND2" miR20a#, miR18a/b#, miR19b/a#, miR16# HMGB1# miR410", miR495" CCNE2# miR34a" HMGB2# miR23a/b" CXCL10" miR16# LMNB1# miR23a/b" CXCL12" miR19b/a# LMNB2# miR24", miR30b" DUSP10" miR20a#, miR92a# PAK1# let7a/f ", miR221" FAS" miR1275# PPP1CC# miR140-5p", miR27b" HMGB2# miR23b" RELB" miR7# IFIT2" miR92a# TNFRSF1B# let7a/f ", miR22", miR338-3p", IGF1" miR18a/b#, miR1275#, miR495" miR19b/a#, miR16# UNG# miR195", miR495" IL1RAP# miR31", miR146a" IRF9"" miR20a# ITGA4" miR20a# ITGAV" miR92a# ITGB3" miR19-b/a# LMNB1# miR23b" MMP2" miR20a# PARP1# miR31" PTGS2" miR16# RAG1" miR92a# RUNX1" miR20a#, miR18a/b# STAT2" miR19-b/a# TGFa" miR7# TIFA# miR181a" TNFRSF1B# miR338-3p" TNFRSF9" miR1275# TNFSF9" miR16#

NOTE: From immune response pathway in HCAEC at 6 h and 24 h after a SD and 6 h and 24 h after MF irradiation. Upregulated target genes and miRNAs are shown in bold.

STAT1 at protein level was confirmed by Western blot to simulate hypofractionated (10 Gy SD) and conven- analysis. P53 and P53-regulated P21, MDM2 proteins were tionally fractionated (2 Gy 5 MF) regimens typically upregulated in response to SD and MF, whereas RAD51 was used for radiotherapy in the clinic. The microarray data downregulated only after MF. CYCLIN D2, CASPASE 1, showed that exposure to MF resulted in more robust and STAT-1 were upregulated after MF (Supplementary changes in gene and miRNA expressions in terms of Fig. S1). number and magnitude, compared with the SD. The MF radiation induced a cohort of mRNAs and miRNAs Discussion associated with stress response, immune response, cell Current radiation therapy techniques expose both nor- cycle, apoptosis, fibrosis, cardiovascular events, and angio- mal tissue and tumors to a wide range of dose size and genesis. Using the ingenuity pathway target filter program, fractionation, with a substantial amount of normal tissue we identified the miRNAs that showed inverse and direct potentially being irradiated (6). This study was undertak- correlations with the differentially expressed target genes en to understand the effect of SD and MF radiation on in response to single and fractionated irradiation. endothelial cells using clinical-relevant schedules to com- Cell cycle and DNA replication/DNA damage stimulus/ plement our recently reported tumor data (6, 8–10, 12). repair were the top gene ontology categories altered in the The radiation protocols for the present study were selected irradiated cells, and the effect was more pronounced in

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Palayoor et al.

cells exposed to MF. Ionizing radiation-induced DNA ing BCL-2 (28), were downregulated, and miR21, which is damage results in activation of various DNA repair path- implicated in suppression of apoptosis (29), was upregulated ways and cell-cycle checkpoints resulting in a temporary suggesting that miRNAs coordinate apoptotic pathway in arrest in cell-cycle progression to allow cells to repair the irradiated HCAEC. Importantly, the upregulation of damaged DNA. If the damage is too severe, cells are FAS by SD and MF may also contribute to apoptosis in the eliminated by apoptosis (14). BRCA1, ATM, and P53 irradiated cells. play key roles in DNA damage response (15–17). The two The irradiated endothelial normal cells showed significant major pathways of DNA damage repair are homologous downregulation of DNA repair genes. Similar results were recombination (HR) and nonhomologous end joining observed in the irradiated LNCaP prostate cancer cells (NHEJ; ref. 18). BRCA1 regulates DNA repair by promot- harboring wild-type p53 (10). This is in contrast to the ing HR in concert with BRCA2 and RAD51, and also response of p53-mutated PC3 prostate cancer cells to radi- inhibits NHEJ to restrict the extent of deletion at the break ation treatment observed in our earlier study (9). Also, no site (17). The present gene expression analysis revealed that significant change in DNA repair genes was observed in the in addition to BRCA1, both BRCA2 and RAD51 from HR irradiated MCF-7, SF539, and DU145 tumor cells, pathway were downregulated in cells exposed to MF. Treat- although MCF7 and SF539 cells express wild-type p53 ment with single and fractionated radiation resulted in the (8). These findings indicate that the expression of DNA upregulation of ATM, and several p53-regulated genes repair genes in response to radiation exposure in tumor cells including MDM2, which in turn controls p53 activity, and is not strictly dependent on the p53 status. In fact, p53- CDKN1A, which regulates cell-cycle checkpoints (19, 20). independent pathways for repair such as P21-PCNA have Cdkn1a, cyclin-dependent kinase (cdk) inhibitor (p21), been reported (30). inhibits cyclin E-cdk2, cyclin D-cdk4, and cyclin A-cdk2 Radiation-induced vascular damage is considered to be complexes (21). The p53-independent check points follow- related to the inflammatory changes in the microvasculature – in vitro in vivo ing ionizing radiation operating at the G2 M transition are (31). Preclinical and studies have demon- mediated by the ATM-Chk1-cdc25C-cyclin B/cdc2 path- strated that ionizing radiation triggers proimmunogenic and way (21). In the present study, many cell-cycle regulatory inflammatory changes in the tumor cells/tumor microenvi- genes including those encoding cyclins CCNB1, CCNE2; ronment, making tumors more susceptible to immunother- kinases CDK1 (CDC2), CDK2; and phosphatases CDC25A, apy (32–36). The ability of radiation to promote the anti- CDC25B, and CDC25C were downregulated. Although tumor immunity has been a subject of great interest, and p53-regulated DNA repair genes DDB2, GADD45a, preclinical studies have reported that the outcome depends DDIT4, and TRIM22 were upregulated, the majority of on the radiation dose and fractionation protocols used (1, 5). DNA repair genes including those encoding DNA poly- Moreover, in 2 mouse tumor models, the combination of merases, primases, and replication factors were downregu- fractionated radiotherapy and anti–CTLA-4 antibody to one lated in the irradiated HCAEC. tumor site induced systemic tumor control as observed by a MiRNAs play an important role in controlling the reg- complete regression in a second palpable tumor outside the ulation of DNA damage response (14). The present study radiation field (abscopal effect; ref. 37). The molecular identified several miRNAs associated with target genes from changes in the irradiated tumor cells that contribute to ATM and P53 signaling pathways and cell-cycle checkpoints immunogenic cell death include degradation of proteins, in the irradiated HCAEC. Although a few microRNAs were release of "danger signals" calreticulin and high mobility differentially regulated in HCAEC by SD such as miR34a, group protein B1 (HMGB1), and ATP which promote miR136, miR140-3p, miR326, miR338-3p, and miR874, priming of antitumor T cells by dendritic cells (33, 36). these miRNAs have been implicated to coordinate the Radiation-induced upregulation of chemokines enhances induction of cell death by apoptosis under various stresses immune cell trafficking to attract activated T cells to the – – (22 26). miR874 has been shown to induce G2 M arrest irradiated tumor site (35). The cancer cells that survive the and cell apoptosis by targeting HDAC1 (25). In agreement radiation insult display enhanced expression of adhesion with these findings, exposure to SD resulted in a reduction in molecules ICAM-1, death receptor Fas, and MHC-1 anti- the percentage of HCAEC in G1 and an increase in cells in gen-presenting molecules, resulting in an improved recog- G2. The surviving fraction of cells exposed to the higher nition and killing by antitumor T cells (38). Interestingly, biologically effective dose regimen SD was much lower than the microarray analysis revealed that several genes from the surviving fraction of cells treated with MF. These data immune response category were differentially expressed in suggest that the majority of cells accumulated in G2 block the irradiated HCAEC. The majority of genes in this following SD exposure did not recover and were eliminated. category were upregulated and the gene expression was more On the contrary, exposure to MF resulted in differential robust in cells exposed to MF. The immune response genes expression of proapoptotic as well as antiapoptotic miRNAs differentially expressed in the irradiated HCAEC included indicating that the final outcome would depend on the genes regulating adhesion molecules, chemokines and cyto- cumulative effect of these opposing miRNAs. For instance, kines, receptors for chemokines, and HLA MHC class I although miR17-92 cluster and miR7, which are shown to and II antigens. There is increasing evidence that miRNAs be inhibitors of apoptosis (27), were reduced in HCAEC by function as an effective system to regulate the magnitude of MF, miR15 and miR16a, which induce apoptosis by target- inflammatory responses (39). Accordingly, many miRNAs

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mRNA and miRNA Proﬁles of Irradiated Endothelial Cells

that activate and dampen the immune response are altered in ofaparticulartypeandasimplemeanstocustomizethis HCAEC exposed to SD or MF, indicating that this process is expression level for each distinct cell type, but also offers a very tightly regulated. For instance, at 24 hours after frac- mechanism to rapidly respond to stress situations. The tionated radiation exposure, the miRNAs from miR17-92 cohort of miRNAs influenced by different regimens of cluster were downregulated and showed inverse correlation radiation indicates this. Although miRNAs expressed in with several of the immune response genes upregulated at response to SD coordinate the induction of immune this time point. miR146 is considered to be a key regulator in response factors and apoptosis, the miRNAs in MF innate as well as adaptive immune responses. Although fine-tune several processes such as DNA repair, fibrosis, miR146a was upregulated by SD and MF in HCAEC, the angiogenesis in addition to immune response and target filter analysis revealed inverse correlation between apoptosis. miR146a and only 2 differentially expressed immune Our previous studies have shown that the tumor cells that response genes. However, CCL5, CXCR4, DDX58, IL1F10, survive MF have substantially different phenotype than the IRAK2, LTB, MR1, and STAT1 showed direct correlation untreated cells or the cells treated with SD and present a with miR146a. The target filter analysis identified several unique opportunity to exploit the radiation-induced changes othermiRNAsshowinginverse correlations with immune to improve cancer therapy (9, 10), with the underlying theme response genes emphasizing the role of miRNAs in of radiation as "focused biology" (46). The gene expression immune response and inflammation. These data suggest profile of endothelial cells in response to fractionated radi- that the irradiated endothelial cells may contribute to ation resembles to that seen in p53 wild-type (cell cycle, radiation-induced immune response during radiation DNA replication/repair) as well as p53-mutated (immune therapy. response) tumor cells observed in our previous studies (9, The activation of growth factor, cytokine, and chemokine 10). Several studies have demonstrated a potential role for cascades in response to the radiation-induced vascular injury radiation as an immunologic adjuvant (1, 5, 32, 33, 36, 38). also contributes to the radiation-induced fibrosis of normal The present study suggests that endothelial cells may con- tissue (40). As mentioned above, several genes regulating tribute to systemic changes during radiotherapy recognizing cytokines and chemokines were upregulated in the irradiated that modern radiation therapy techniques can be used to endothelial cells. Among all radiation-induced cytokines, target tumors and reduce normal tissue hot spots, but at the TGFb activation is of particular relevance, as it elicits strong same time more normal tissue, and thus endothelial cells, and long-lasting microenvironmental changes (7, 41). receive some dose. TGFb plays a central role in fibrosis by stimulating produc- The importance of the tumor microenvironment is greatly tion of new matrix proteins such as fibronectin, collagens, emphasized in cancer therapy. Tumors are complex struc- and proteoglycanes (7, 42). The present gene expression tures with the stroma and infiltrating cells impacting tumor analysis showed an increase in TGFB as well as COL1A2 and survival and progression, and the acquired ability for epi- FBN1 at 6 and 24 hours after MF. The upregulation of thelial–mesenchymal transition (47). Radiotherapy signifi- COL1A2 inversely correlated with the downregulation of cantly alters tumor microenvironment. Certainly, normal miR7 and miR92a at 24-hour time point. FBN1 showed tissue effects of radiation depend on changes to parenchyma inverse correlation with miR1275 and miR92a. Previous cells that are organ-specific and also to endothelial cells that studies showed that upregulation of collagens and fibrillin 1 are ubiquitous. Recent studies indicate that tumor-derived in the regions adjacent to infarct during remodeling after endothelial cells differ from normal endothelial cells at both myocardial infarction is regulated by downregulation of functional and molecular levels, and endothelial cells derived miR29 (43). Postinfarct cardiac fibrosis on the other hand from different tumors are shown to be divergent dependent was inhibited by forced expression of miR101 (44). Inter- on the origin of the tumor (48). Although these differences estingly, miR29 blocks fibrosis by inhibiting the expression remain to be better understood and exploited, dissecting out of extracellular matrix components, whereas miR21 pro- the contributions of the various tissue components to motes fibrosis in SMCs after vascular injury by stimulating radiation response is necessary to best understand the aggre- MAPK signaling (45). In the irradiated HCAEC, miR29, gate picture. The present data warrant further investigations miR21, and miR101 were upregulated, whereas miR7 and on radiation response in both normal as well as tumor- 92a were downregulated. These observations indicate that derived endothelial cells. induction of fibrosis is also a balance between the actions of Improving the therapeutic ratio is critical to effective these miRNAs, in agreement with the mechanism of action clinical radiotherapy and treatment in general. In our current of miRNAs acting as rheostats to fine-tune and modulate the focus on understanding and targeting the cells that survive outcome based on the intensity of damage (11). MF (6), the changes in endothelial cells would be of strategic As seen with the HCAEC, the miRNA-based gene- importance. Furthermore, it may be possible to use changes regulatory system provides a flexible and conditional induced by the endothelial cells including factors found in option that would be particularly useful when mRNA the blood to understand how the tumor is responding and expression must be fine-tuned to different levels in dif- use this information for immunotherapy or other molecu- ferent cell types. The posttranscriptional dampening of larly targeted treatments. Although much remains to be gene expression by miRNAs not only offers both a done, having normal tissue data facilitate developing better mechanism for more uniform gene expression for cells and improved therapeutic approaches.

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Disclosure of Potential Conflicts of Interest Acknowledgments M.T. Falduto is chief technology officer and has ownership interest in GenUs The authors thank Dr. T. Adilakshmi for critical reading of the article BioSystems, Inc. S.R. Magnuson is president and has ownership interest in GenUs and editorial help, Dr. Charles B. Simone II (Department of Radiation BioSystems, Inc. No potential conflicts of interest were disclosed by the other authors. Oncology, Hospital of the University of Pennsylvania, Philadelphia, PA) for his expert advice for application of clinical radiotherapy regimens in the Authors' Contributions laboratory settings. Conception and design: S.T. Palayoor, M. John-Aryankalayil, C.N. Coleman Development of methodology: S.T. Palayoor, M. John-Aryankalayil, M.T. Falduto, S.R. Magnuson, C.N. Coleman Acquisition of data (provided animals, acquired and managed patients, provided Grant Support facilities, etc.): M. John-Aryankalayil, A.Y. Makinde, M.T. Falduto, S.R. Magnuson This work was supported by the Intramural Research Program of the Center for Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- Cancer Research, NCI, NIH. The costs of publication of this article were defrayed in part by the payment of page tational analysis): S.T. Palayoor, M. John-Aryankalayil, M.T. Falduto, C.N. Coleman advertisement Writing, review, and/or revision of the manuscript: S.T. Palayoor, M. John- charges. This article must therefore be hereby marked in accordance with Aryankalayil, A.Y. Makinde, M.T. Falduto, C.N. Coleman 18 U.S.C. Section 1734 solely to indicate this fact. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.T. Palayoor, M. John-Aryankalayil, A.Y. Makinde, M.T. Falduto, S.R. Magnuson Received November 26, 2013; revised April 1, 2014; accepted April 2, 2014; Study supervision: S.T. Palayoor, C.N. Coleman published OnlineFirst April 30, 2014.

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www.aacrjournals.org Mol Cancer Res; 12(7) July 2014 1015

Differential Expression of Stress and Immune Response Pathway Transcripts and miRNAs in Normal Human Endothelial Cells Subjected to Fractionated or Single-Dose Radiation

Sanjeewani T. Palayoor, Molykutty John-Aryankalayil, Adeola Y. Makinde, et al.

Mol Cancer Res 2014;12:1002-1015. Published OnlineFirst April 30, 2014.

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