Research Article Genetic Background Modulates ...

Hindawi Publishing Corporation International Journal of Genomics Volume 2015, Article ID 461038, 7 pages http://dx.doi.org/10.1155/2015/461038

Research Article Genetic Background Modulates lncRNA-Coordinated Tissue Response to Low Dose Ionizing Radiation Jonathan Tang, Yurong Huang, David H. Nguyen, Sylvain V. Costes, Antoine M. Snijders, and Jian-Hua Mao Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road MS977, Berkeley, CA 94720, USA Correspondence should be addressed to Antoine M. Snijders; [email protected] and Jian-Hua Mao; [email protected] Received 30 October 2014; Revised 4 February 2015; Accepted 4 February 2015 Academic Editor: Elena Pasyukova Copyright © 2015 Jonathan Tang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Long noncoding RNAs (lncRNAs) are emerging as key regulators of diverse cell functions and processes. However, the relevance of lncRNAs in the cell and tissue response to ionizing radiation has not yet been characterized. Here we used microarray profiling to determine lncRNA and mRNA expression in mammary glands of BALB/c and SPRET/EiJ mice after low-dose ionizing radiation (LDIR) exposure. We found that unirradiated mammary tissues of these strains differed significantly in baseline expressions of 290 lncRNAs. LDIR exposure (10 cGy) induced a significant change in the expression of many lncRNAs. The vast majority of lncRNAs identified to be differentially expressed after LDIR in either BALB/c or SPRET/EiJ had a significantly correlated expression pattern with at least one LDIR responsive mRNA. Functional analysis revealed that the response to LDIR in BALB/c mice is highly dynamic with enrichment for genes involved in tissue injury, inflammatory responses, and mammary gland development at 2, 4, and 8 weeks after LDIR, respectively. Our study demonstrates that genetic background strongly influences the expression of lncRNAs and their response to radiation and that lncRNAs may coordinate the tissue response to LDIR exposure via regulation of coding mRNAs.

1. Introduction Ionizing radiation is a well-known carcinogen in humans, and breast is one of the most sensitive organs to radiogenic cancer [1]. The rate of breast cancer in postwar Japan was among the lowest in the world, but breast cancer contributed a disproportionately large fraction of the radiation-related cancer burden in atomic bomb survivors [2, 3]. The data from the Hiroshima and Nagasaki survivors provides strong evidence for increased breast cancer following single acute doses of 20 cGy and linearity with increasing dose [3–6]. Also, an increase in the incidence of breast cancer has been observed in areas affected by the Chernobyl accident, which resulted in radioactive contamination of large areas of Belarus and Ukraine [7]. A twofold increase in risk was observed when comparing the most (>40 mSv cumulative dose) and least contaminated regions. Interestingly, the increase appeared 10 years after exposure and was most prominent in women exposed at younger age. More than 50,000 women in the United States have been treated with chest radiation (≥20 Gy) for a pediatric or young

adult cancer. Children treated from cancer with radiotherapy have a 2.9 relative risk of subsequent malignancy compared to those who were not [8, 9]. A systematic review of 14 studies concluded that risk of breast cancer increased as early as 8 years following chest radiation and did not plateau with increasing length of follow-up [10]. Studies estimating lowdose radiation-induced cancer risk from diagnostic X-rays and CT scans have found a small but significant increased lifetime risk [11, 12]. While the benefits of diagnostic X-ray and CT scans outweigh potential individual lifetime risk, their use should be justified and alternatives considered. We know remarkably little of molecular mechanisms that may be protective or risky for breast cancer after exposure to low-dose ionizing radiation (LDIR). Identification of transcriptomic changes induced by LDIR in mammary tissue will be valuable to elucidate the molecular mechanisms associated with radiation-induced breast cancer. Long noncoding RNAs (lncRNAs), which initially were thought of as transcriptional noise, are emerging as key regulators of a multitude of cellular processes by taking part in epigenetic, transcriptional, and posttranscriptional regulation of gene expression [13, 14].

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International Journal of Genomics Coding RNA BALB/c SPRET IL4R

NCF2

NCF1

IFNAR1

ITGA1

Trk receptor HRAS

PI3K (complex) Shc

RAPSN

Laminin

TNK2 GRB2

CDC25B

Phosphatase EYA4

LPAR5 CD44

PTPN12 Focal adhesion kinase

RXFP4 RAMP3∗

OPN1LW Gpcr

GPRC5B

Chemokine

PTPase PTP4A1 ARHGAP4

SH2B3 CXCL12 PTPDC1

CYSLTR2 EDNRA

CKLF

DDX39B

KIN MXD1

lncRNA SPRET BALB/c

MISP

SMC3 PCNA

Mediator

TIMELESS

RPS25

RPA Ribosomal 40s subunit

DCLRE1C

PAX2

C17orf96 CycIin D

ATM

Jnk

RPSA∗

POLR2H

DNA-directed RNA polymerase POLR2L

CRK

RMI1 RPS3

POLR1B

Creb Akt

BALB/c

PDGFRB

SPRET

CSF2RB

ARRB2

FANCF

−1.50 −1.00 −0.50 0.00 0.50 1.00 1.50

−1.50 −1.00 −0.50 0.00 0.50 1.00 1.50

IGF1

(a)

(b)

STAT5a/b

PES1 PHLPP1

(c)

Figure 1: Significant strain differences in lncRNA and coding RNA expression in mammary tissues of BALB/c and SPRET/EiJ mice. (a)-(b) Hierarchical clustering of baseline differences in lncRNA (a) and coding RNA (b) expression in mammary gland tissues of 8-9-week-old BALB/c and SPRET/EiJ mice (fold-change 1.5; 𝑝 value < 0.001). Increased expression indicated in red and decreased expression in green. (c) Gene interaction networks of genes expressed at lower levels in BALB/c mammary tissues when compared to SPRET/EiJ (top) and genes expressed at higher levels in BALB/c compared to SPRET/EiJ (bottom) (see Figure S3 for annotation of molecular shapes used in the networks).

The lncRNAs have a weaker evolutionary constraint and lower levels of expression compared to the protein-coding transcripts [15, 16] but exhibit more tissue specific expression than the protein-coding genes. Recently, a number of studies have shown that lncRNA expression can be deregulated in human cancers [17, 18]. As the functions of individual lncRNAs in cancer are beginning to be elucidated, they are being categorized and referred to as either tumor suppressor or oncogenic lncRNAs, in the same way as traditional proteincoding cancer genes. However, the relevance of lncRNAs in the cell and tissue response to ionizing radiation has not yet been characterized. In this study, we used Agilent SurePrint G3 microarrays to profile lncRNA and mRNA from mammary glands of BALB/c mice 2, 4, and 8 weeks after irradiation and of SPRET/EiJ mice 4 weeks after irradiation with 10 cGy of X-radiation. We identified lncRNA and mRNA expression signatures for each time point after irradiation in comparison to sham. Of the total 1338 lncRNAs identified to be differentially expressed after LDIR in either BALB/c or SPRET/EiJ, 1337 had

a significantly correlated expression pattern with at least one mRNA that was also differentially expressed after LDIR. Our results indicate lncRNAs may exert a partial or key role in the regulation of coding RNA expression induced by radiation.

2. Materials and Methods 2.1. Mice and Irradiation. BALB/c and SPRET/EiJ mice were purchased from Jackson Laboratory, housed four per cage under a 12 hr light and 12 hr dark cycle, and fed with Lab Diet 5008 chow and water ad libitum. The mice were irradiated whole body at 8-9 weeks of age to a single dose of 10 cGy using a Precision X-ray Inc RAD320 320 kVp Xray machine, operated at 300 kV, 2 mA. Mammary tissues were collected for gene expression profile at 2, 4, and 8 weeks after irradiation. All animal experiments were performed at Lawrence Berkeley National Laboratory and the study was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was approved by the Animal

International Journal of Genomics Welfare and Research Committee of the Lawrence Berkeley National Laboratory. 2.2. Expression Profile by Microarray. Total RNA quality and quantity were determined using Agilent 2100 Bioanalyzer and NanoDrop ND-1000. Agilent SurePrint G3 Mouse GE 8x60K Microarrays were used according to the manufacturer’s protocol (arrays contained 39,430 Entrez gene RNAs and 16,251 lncRNAs). All processes were done by Ambry Genetics (Aliso Viejo, CA). Microarray data have been deposited at NCBI GEO (accession number: GSE62662). 2.3. Data Analysis. Data normalization was performed with GeneSpring GX12.5 (Agilent Technologies). Signal intensities for each probe were normalized to the 75th percentile without baseline transformation. Genes that were differentially expressed between sham and irradiated were identified by the unpaired Student’s 𝑡-test with a 𝑝 value cut-off of 0.05 (𝑝 value < 0.001 for baseline strain comparison) and a fold-change criteria of more than 1.5. 2.4. Correlation Analysis. For each of the 8 experimental treatment groups, the average expression values of the 3 biological replicates were first calculated for each mRNA and each lncRNA. Significantly correlated pairs of mRNA and lncRNA were calculated using a standard permutation test [19]. In brief, for each potential mRNA and lncRNA pair, the 8 mRNA values were randomly rearranged and a correlation coefficient was calculated between the 8 mRNAs and 8 lncRNAs values. Permutations were repeated 10,000 times to derive a distribution of 10,000 correlation coefficients. The 𝑝 value reported in this work represents the percentage of permutations leading to a higher correlation than the original correlation between the 8 mRNAs and 8 lncRNAs values. In other words, the lower the 𝑝 value is, the more likely the mRNA and lncRNA pair is not randomly associated. 2.5. Functional Analysis. Gene lists were annotated with biological functions using Ingenuity Pathway Analysis (IPA), KEGG pathway analysis (http://bioinfo.vanderbilt.edu/webgestalt/) and DAVID GO gene ontology (http://david.abcc .ncifcrf.gov/; 𝑝 ≤ 0.05). Annotations for the various shapes used in the IPA networks in Figures 1–3 are shown in Figure S3 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/461038. lncRNA and mRNA correlation networks were generated using Cytoscape.

3. Results and Discussion 3.1. Differential Expression of lncRNA and mRNA between BALB/c and SPRET/EiJ Mammary Tissues. To identify potential lncRNAs and mRNAs that may determine susceptibility to radiation-induced breast cancer, we profiled two inbred strains of mice with differing genetic susceptibilities: BALB/c mice as more sensitive and SPRET/EiJ as more resistant. BALB/c mice carry two DNA-PKcs polymorphisms with reduced catalytic subunit activity and defective nonhomologous-end-joining of double strand breaks [20]. We identified 195 lncRNAs as upregulated and 95 lncRNAs

3 as downregulated in BALB/c in comparison to SPRET/EiJ (fold-change 1.5; 𝑝 value < 0.001) (Figure 1(a); Table S1). Additionally, 582 mRNAs were upregulated and 402 mRNAs were downregulated in BALB/c in comparison to SPRET/EiJ (fold-change 1.5; 𝑝 value < 0.001) (Figure 1(b); Table S1). Gene ontology analyses of differentially expressed genes between BALB/c and SPRET/EiJ showed significant enrichment for metabolic processes (𝑝 = 5.9𝐸 − 06), ion binding (𝑝 = 3.00𝐸 − 08), and chemokine signaling (𝑝 = 0.02) (Figure 1(c); Table S2). Our analyses identified significant strain differences in gene expression between BALB/c and SPRET/EiJ mammary tissues. We found significant differences in the expression of a number of chemokines including CXCL10, CCL6, and CCL25, which were expressed at higher levels in mammary tissues of the more sensitive and susceptible BALB/c mice and which have previously been associated with breast cancer progression when over expressed [21]. 3.2. lncRNA and mRNA Expression Signatures of Irradiated Mammary Tissues. To identify lncRNA and mRNA expression changes induced by low-dose ionizing radiation (LDIR), we profiled lncRNA and mRNA expression from mammary glands of BALB/c mice 2, 4, and 8 weeks after irradiation and of SPRET/EiJ mice 4 weeks after irradiation with 10 cGy of X-radiation. For BALB/c mice, a total of 357, 480, and 335 lncRNAs and 550, 911, and 389 coding RNAs were identified to be differentially expressed at weeks 2, 4, and 8 after IR in comparison to sham (fold-change 1.5; 𝑝 value < 0.05), respectively (Figure 2(a); Table S1). For SPRET/EiJ, a total of 327 lncRNAs and 424 mRNAs were identified as differentially expressed at week 4 after irradiation in comparison to sham (fold-change 1.5; 𝑝 value < 0.05) (Figure 3(a); Table S1). Few coding-RNAs and lncRNAs were found to be differentially expressed at different time points (Figure S1(A) and S1(B)) and between BALB/c and SPRET/EiJ (Figure S1(C)). To determine the biological functions associated with the LDIR response, we excluded genes whose levels fluctuate in the mouse mammary gland across the estrous cycle. We recently mapped transcript-level changes across the estrous cycle in the murine mammary gland using RNA sequencing and defined a comprehensive estrous variable gene signature of 3893 genes whose levels fluctuate in mammary glands of BALB/c mice [22]. Comparison of our mapped LDIR genes in mammary glands of BALB/c and SPRET/EiJ mice with the estrous signature revealed an approximate 20% overlap in BALB/c (Figure 2(b)) and 9% overlap in SPRET/EiJ mice (Figure 3(b)). Nonoverlapping and differentially expressed LDIR genes for each of the time points were then computationally mapped to biological functions, pathways, upstream regulators, and networks. These analyses suggested that the LDIR response signatures in mammary glands of BALB/c mice transitions between time points and is distinct from the LDIR response in SPRET/EiJ mice. Two weeks after LDIR exposure pathways and biological functions significantly enriched in mammary glands of BALB/c mice compared to sham irradiated mice included chemokine signaling (𝑝 = 0.01), CCR3 signaling in

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International Journal of Genomics Coding RNA Sham LDIR

lncRNA Sham LDIR

TFAP2D AGAP2

Estrous signature

BALB/c LDIR

MAGI2 KMT2D MAPK14

Oxct2a/Oxct2b UXS1

ICOSLG/LOC102723996 SLC4A5

Akt

KRT6B Cytokeratin WIPI1

2 weeks

CREM Hsp70 Hsp27 ESR1 PCSK1

3811

82

GLYCTK

ZBTB7A

354

Actin

RBFOX2 KRT1

KLK3

Ap1

IGHA1 F Actin

UTRN

KLF1

Cyp2d9 (includes others)

TNC PA2G4 CAPZA1 GABPB1

Tmsb4x (includes others)

FGG

PDK1

MPP1

AGBL2

RNA polymerase II

KCNAB2

SLC27A2 Akt

TP63

ULK2

4 weeks

KCNA6 ENO3

Hsp90 Hsp70 TCF7L2

HIPK2 PCF11

3719

174

552

PAX2

EGLN3

DNAJB8

CYP2E1

HCFC1

HSP

HSPA4

IGHA1 FAN1

TNFRSF13C POU2AF1 AREG

CHF Cyclin A/Cdk2

8 weeks

GREB1 AMD1 WNT4 Cyclin A PRL

58

1.50

1.00

0.50

0.00

−0.50

−1.00

−1.50

3835

(a)

(b)

250

TRERF1

C15orf39

PHF20L1 CRYAA/LOC102724652 CRYBB1

AKAP4 SUMO2

PGR

CCNA1

ATF1 CCK CDK1-Cyclin A

SYNPO Histone h3 PDC PML Hsp90 PTCH1 FKBP51-TEBP-GRHsf4 HSP90-HSP70 RPRM CCNT1 ERMAP PIH1D3 Hsp70 RNA polymerase II TSACC CENPW Dnajb1-Hsp70 Mucl1/Spt1 HSPA4 POLR2J2/POLR2J3 PPP1R15A SYT4

(c)

Figure 2: Significant lncRNA and coding RNA expression changes after LDIR exposure in mammary tissues of BALB/c mice. (a) Hierarchical clustering of lncRNAs (left) and coding RNAs (right) differentially expressed (fold-change 1.5; 𝑝 < 0.05) in mammary gland tissues of BALB/c mice at 2, 4, and 8 weeks after sham or LDIR exposure. Increased expression indicated in red and decreased expression in green. (b) Comparison of genes differentially expressed after low-dose radiation at each timepoint with a gene signature containing genes whose expression significantly changes in the mammary gland across the female estrous cycle. (c) The most significant gene interaction networks of estrous cycle independent genes differentially expressed after LDIR at 2, 4, and 8 weeks after exposure (see Figure S3 for annotation of molecular shapes used in the networks).

eosinophils (𝑝 = 0.05), cellular movement (3.92𝐸 − 04 < 𝑝 < 3.41𝐸 − 02), and cell death and survival (1.29𝐸 − 03 < 𝑝 < 3.41𝐸 − 02). Gene interaction networks were enriched for tissue and endocrine system injury (Figure 2(c) top panel) and significant predicted upstream regulators (Table S3) include GLI2 (𝑝 = 5.29𝐸 − 04) and SATB1 (𝑝 = 1.98𝐸 − 03). Similar to the two-week LDIR response, GLI2 was predicted to be an upstream regulator (𝑝 = 4.52𝐸 − 03; Table S3). GATA3 and STAT6 were among other significant upstream regulators associated with the four-week low-dose response (Table S3). We furthermore observed that the mammary gland of BALB/c mice four weeks after LDIR was enriched for inflammatory response genes (1.84𝐸 − 05 < 𝑝 < 1.63𝐸 − 02), cell movement (3.51𝐸 − 06 < 𝑝 < 1.45𝐸 − 02), cell-cell

signaling (2.79𝐸 − 05 < 𝑝 < 1.63𝐸 − 02), and morphology (1.41𝐸 − 04 < 𝑝 < 1.60𝐸 − 02), while gene interaction networks were enriched for lipid metabolism (Figure 2(c) middle panel). Interestingly, similar responses were observed in mammary glands of SPRET mice at 4 weeks after LDIR including inflammatory response functions (1.69𝐸−03 < 𝑝 < 4.49𝐸 − 02), cell-cell signaling (5.54𝐸 − 04 < 𝑝 < 4.49𝐸 − 02), and morphology (8.31𝐸 − 05 < 𝑝 < 4.49𝐸 − 02), suggesting that the functional response is similar across strains and is independent of the gene transcript response. At 8 weeks after LDIR we observed downregulation of genes involved in mammary gland development including progesterone receptor, prolactin, amphiregulin, and WNT4 (Figure 2(c) bottom panel; Table S3).

International Journal of Genomics

5

Coding RNA Sham LDIR

lncRNA Sham LDIR

IL1RN

4 weeks

CSDC2

CA12

RNA polymerase II

Estrous signature

SPRET LDIR

CXCL3 NF𝜅B (complex)

Mug1/Mug2 KLK3 MED1 CDK1

RPS6KA3

CFLAR

CREM Histone h3

41

315

NAB2 ERK

WDR61 GNAL

1.50

1.00

0.50

0.00

−0.50

−1.00

−1.50

MAPK4

(a)

KIF1A

Histone h4

EGR4

3852

IFNA4

IRF1

DCX

SNTG1

CEACAM1 F Actin

Calmodulin

DMD

Actin

VSNL1

TBX5 Tropomyosin TNNT3

(b)

(c)

Figure 3: Significant lncRNA and coding RNA expression changes after LDIR exposure in mammary tissues of SPRET/EiJ mice. (a) Hierarchical clustering of lncRNAs (left) and coding RNAs (right) differentially expressed (fold-change 1.5; 𝑝 < 0.05) in mammary gland tissues of SPRET/EiJ mice at 4 weeks after sham or LDIR exposure. Increased expression indicated in red and decreased expression in green. (b) Comparison of genes differentially expressed after low-dose radiation with a gene signature containing genes whose expression significantly changes in the mammary gland across the female estrous cycle. (c) The most significant gene interaction network of estrous cycle independent genes differentially expressed after LDIR (see Figure S3 for annotation of molecular shapes used in the networks).

3.3. Significantly Correlated lncRNA and mRNA Expression Patterns. To identify lncRNAs potentially regulating the expression of coding RNAs in response to radiation, correlation coefficients on expression data were calculated for each coding RNA and lncRNA that were identified as differentially expressed after LDIR. A permutation-based algorithm was then used to determine which correlations were statistically significant (𝑝 < 0.05; Table S4). We observed that nearly all LDIR modulated lncRNAs were correlated with at least one of the LDIR modulated coding mRNAs (Figure 4(a)). To determine whether these correlations were driven by estrous variations, we only considered genes whose expression levels were not overlapping with our previously determined estrous signature (Figure 2(b)). Again, we observed that nearly all differentially expressed lncRNAs were correlated with at least one differentially expressed mRNA suggesting that estrous cycling does not affect the strong correlation between lncRNA and mRNA expression after LDIR. To test the robustness of these correlations, we compared the number of lncRNAs associated with at least one mRNA at three different 𝑝 values (𝑝 < 5𝐸 − 02, 𝑝 < 5𝐸 − 03, and 𝑝 < 5𝐸 − 04). At 𝑝 < 5𝐸 − 02 or 𝑝 < 5𝐸 − 03, nearly all (97–100%) differentially expressed lncRNAs were found to be correlated with at least one mRNA (Table S5). At 𝑝 < 5𝐸−04, corresponding to a correlation coefficient >0.9, we still observed a significant fraction (63–81%; Table S5) of lncRNAs correlated with mRNAs. Representative correlation networks of lncRNAs are shown in

Figure 4(b) (𝑝 < 5𝐸 − 03) for each of the timepoints. We furthermore observed that the same lncRNA correlates with different gene sets across different time points (Figure S2). Taken together these data show that LDIR induces coordinated changes in lncRNA and mRNAs and suggests a critical role for lncRNAs in mediating the low-dose radiation response.

4. Conclusions In this study, we demonstrate that genetic background strongly influences the expression of lncRNAs and their response to low-dose radiation by transcriptomic analysis of mouse mammary glands using microarrays that contain both lncRNAs and coding RNAs. We have identified a number of lncRNAs that are significantly changed after exposure to LDIR at three different timepoints after radiation exposure. These lncRNAs have the potential to be surrogate indicators of tissue radiation responses. Moreover, the changes in the expression of lncRNAs are significantly correlated with the expression of coding RNAs, suggesting that lncRNAs may coordinate the tissue response to radiation via regulation of coding mRNAs. However, the specific regulatory mechanism of this control requires further investigation, and knock-out and overexpression of the lncRNA genes in mice and other model systems should be performed to increase our understanding of the regulatory mechanisms in response to LDIR.

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International Journal of Genomics BALB/c 2 weeks after LDIR

KIAA1549 A930038B10Rik

CNTLN

4930474G06Rik

MPP1 1700007E05Rik

Krtap4-1/Krtap4-2

FAM151A

Cyb5r3

Usp17la

TACR2

MAPK14 Cyp2d9 MAN2A2 KMT2D

Coding RNA

SLC5A11

A 30 P01024556

A 30 P01025227 ATP2B2 ANXA13

SLC17A5 UTRN

Vmn2r88

EFHB

HSF5

ARL4D

LOC102633216

ETV4 G6pd2 C9orf91

4933422A05Rik AW047481

1700034I23Rik/Gm10731 POP5

SGK494

Gm5334

KRT222

A 30 P01030975 HECTD4

PDZRN3

TOB2

SMO

Cyp2d22

E230024E03Rik

A 30 P01030601 KNSTRN

LOC100125594

CFAP97

Olfr1161

PPWD1

OPRM1

ADGB

EGLN1

SLC4A5

Ppap2a

SNAP23

CAPZA1

Vmn2r32

TMC2 PI4KB A 30 P01021708

RBFOX2

C5orf49

KRT1

PIGN

PABPC4L

DOPEY1

A930004J17Rik HELLS

Iglv1

ARMC3

CDH13

GLYCTK

BALB/c 4 weeks after LDIR

KCNA6 3830403N18Rik/Xlr

TMTC2

ZDHHC22 Ang3 GAPT Oxct2a/Oxct2b KCNH8 Krt83 AHNAK2 Tas2r143 ALOX5AP Ear2 C10orf90 SERPINB6 TRRAP RGP1 CRYAA/ MPZL2 RNASE2 LOC102724652 MAGEB18 TUBAL3 SLC7A13 RHOJ ZBTB12 KRTAP12-2 FGB Tmsb4x CALCR SEZ6 Ccr1l1 FGG LOC102640292/ 1700001J03Rik LOC102640399 BCL6B ULK4 EVA1C Gm6760 HTRA1 D630010B17Rik CPA1 FCER1G A 30 P01022631 CXCR1 PPP2R2C ATAT1 A 30 P01021768 TPTE2 Spink11/Spinkl Ear12 FAN1 SLC45A1 Gm4836 Serpinb6e Gm4862 Olfr706 BC030307 Cbr2 MAGIX PCF11 ZNF768 1700009N14Rik SLC27A2 Igk Olfr247 ZNF705A STXBP6 EMR1 MAP3K8 Serpinb6c RNASE12 C12orf60 ADAMTS2 Gm14135 Vmn1r188 C2CD2L ARL11 Gm10941 TFPI CYP2R1 CYSLTR2 DHX34 NR2F1 C3orf18 4933417E11Rik Cmtm2a FGF11 LYPD4 LPO CYP2E1

PPFIBP2

Coding RNA

RAB31

BALB/c 8 weeks after LDIR

Gm10440 SOX21 RALYL

NPY1R

Far2os2

KCNE3

Moxd2

Gm20736 Pantr1 ANKRD34A A 30 P01033122 Ang2 Olfr987/ PABPC5 Olfr988 AKR1C4 Slc13a2os Gm14459 C1orf185 Gm6432 PLA2G2C Vmn1r188 G6PC2 HOXD13 CCNA1 GPAT2 PROKR2 ADAMTS20 NSUN3 KRTAP2-3/KRTAP2-4 ZSCAN10TXNDC8 HPD A 30 P01029234 C430002N11Rik 4930543N07Rik FREM3 LOC102641662 9630041G16Rik A 30 P01020129 Olfr119/Olfr121 PRUNE2 PDE11A Lrrc31 CCK KCNH4 NWD2 LOC101056131 Mrgpra8 TRAPPC3L Gm6961 HIST1H2AK LOC102636289

Coding RNA

SLC26A7

OR13A1

Olfr889/Olfr890

CDK1 C330024D21Rik

Coding RNA

Cdrt4os1

KLK3 Gm7167

OCA2

A830011I04

Celrr

HIST1H2BO

LOC102632061 Rhox5

SPRET 4 weeks after LDIR

Mug1/Mug2

SPOCK1

MED1

A 30 P01026239

TMCO5A

AA623943/Gm11546

A 30 P01027448

OR4C45

Fmr1nb

Btnl5-ps Gm9767 SYCP2L

PROM2

ACIN1

A 30 P01021129

Gm16441/Gm6038

(a)

BAI3

CCDC89 ENTPD7

LOC101928991

AGTPBP1 ANKRD16

TMEM184A

MS4A4A

A 30 P01020817

RABGEF1

9130221J18Rik

1700024P04Rik CEACAM1

LINC RNA

HOXD3

Gm3002

TLX1 C8A

TIGD4

Far2os2

1700084D21Rik OR13G1

CFLAR

2610020C07Rik 1700091H14Rik

A 30 P01024025

BZW2

APOL2

FAM135B

KIAA2026

TRIM45

Dynap TTC25

(b)

Figure 4: Correlated lncRNA and coding RNA expression in mammary tissues after low-dose radiation exposure. (a) Correlation graphs of the average expression values from each experimental treatment group for each mRNA and lncRNA differentially expressed after LDIR at each of the four timepoints. Positive and negative correlations are indicated in blue and yellow, respectively. (b) Representative examples of networks of lncRNAs (purple) significantly correlated in expression with mRNAs (pink).

Conflict of Interests

Authors’ Contributions

The authors declare that there is no conflict of interests regarding the publication of this paper.

Jian-Hua Mao and Antoine M. Snijders provided conception and overall experimental design. Yurong Huang and David

International Journal of Genomics H. Nguyen performed experiments. Jonathan Tang, Sylvain V. Costes, Antoine M. Snijders, and Jian-Hua Mao analyzed microarray data. Jonathan Tang, Antoine M. Snijders, and Jian-Hua Mao prepared all figures and wrote the paper. All authors contributed to paper editing.

Acknowledgment This work was supported by Low Dose SFA Program of the Director, Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract no. DE-AC02-05CH11231.

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