Distinctive microRNA expression signatures in proton-irradiatedmice

Shahid Yar Khan • Muhammad Akram Tariq • James Patrick Perrott •

Christopher Drew Brumbaugh • Hyunsung John Kim •

Muhammad Imran Shabbir • Govindarajan T. Ramesh •

Nader Pourmand

Received: 16 April 2013 / Accepted: 14 June 2013 / Published online: 2 July 2013

� Springer Science+Business Media New York 2013

Abstract Proton particles comprise the most abundant

ionizing radiation (IR) in outer space. These high energy

particles are known to cause frequent double- and single-

stranded DNA lesions that can lead to cancer and tumor

formation. Understanding the mechanism of cellular

response to proton-derived IR is vital for determining

health risks to astronauts during space missions. Our

understanding of the consequences of these high energy

charged particles on microRNA (miRNA) regulation is still

in infancy. miRNAs are non-coding, single-stranded RNAs

of *22 nucleotides that constitute a novel class of gene

regulators. They regulate diverse biological processes, and

each miRNA can control hundreds of gene targets. To

investigate the effect of proton radiation on these master

regulators, we examined the miRNA expression in selected

mice organs that had been exposed to whole-body proton

irradiation (2 Gy), and compared this to control mice (0 Gy

exposure). RNA was isolated from three tissues (testis,

brain, and liver) from treated and control mice and sub-

jected to high-throughput small RNA sequencing. Bioin-

formatics analysis of small RNA sequencing data revealed

dysregulation of (p \ 0.05; 20 up- and 10 down-regulated)

14 mouse testis, 8 liver, and 8 brain miRNAs. The statis-

tically significant and unique miRNA expression pattern

found among three different proton-treated mouse tissues

indicates a tissue-specific response to proton radiation. In

addition to known miRNAs, sequencing revealed differ-

ential expression of 11 miRNAs in proton-irradiated mice

that have not been previously reported in association with

radiation exposure and cancer. The dysregulation of miR-

NAs on exposure to proton radiation suggest a possible

mechanism of proton particles involvement in the onset of

cell tumorgenesis. In summary, we have established that

specific miRNAs are vulnerable to proton radiation, that

such differential expression profile may depend upon the

tissue, and that there are more miRNAs affected by proton

radiation than have been previously observed.

Keywords miRNA � Differential expression �Proton radiation � Mice

Introduction

The space radiation environment consists mainly of trap-

ped particle radiation, solar particle radiation, galactic

cosmic radiation (GCR), and also ionizing radiation (IR).

IR is a well-known modality used in cancer treatment that

also induces oxidative genotoxic stress. It is known that IR

causes cellular damage by either directly damaging DNA,

or by the formation of free radicals [1]. Among IR, protons

S. Y. Khan � M. A. Tariq � J. P. Perrott �C. D. Brumbaugh � H. J. Kim � M. I. Shabbir �N. Pourmand (&)

Department of Biomolecular Engineering, University

of California Santa Cruz, Santa Cruz, CA 95064, USA

e-mail: shahidyarkhan@hotmail.com; pourmand@soe.ucsc.edu

M. A. Tariq

Center for Bionanotechnology and Environmental Research,

Texas Southern University, Houston, TX 77004, USA

Present Address:

M. I. Shabbir

Department of Bioinformatics and Biotechnology, Faculty

of Basic Applied Sciences, International Islamic University,

Sector H10, Islamabad, Pakistan

G. T. Ramesh

Molecular Toxicology Laboratory, Center for Biotechnology

and Biomedical Sciences, Norfolk State University,

700 Park Avenue, Norfolk, VA 23504, USA

123

Mol Cell Biochem (2013) 382:225–235

DOI 10.1007/s11010-013-1738-z

particles represent the largest fraction (85 %) of GCRs and

the understanding of the biological effects of high energy

protons is important in the neoplastic transformation that

can lead to cancer. A limited number of studies have been

performed to evaluate the impact of proton radiations on

the induction of neoplastic transformation in biological

systems. Recently, Sorokina and co-authors studied the role

of proton and c-radiation in the formation of double-

stranded breaks (DSB). They showed that protons and

X-rays at 2 Gy dose rate induced very similar number of c-

H2AX and 53BP1 foci in human umbilical vein cells [2]

and revealed that the biological effects of proton radiation

are similar to the effects of c-radiation and other low-linear

energy transfer (LET) radiation [2]. In another study, HeLa

6 skin fibroblast human hybrid cells were used for the

estimation of neoplastic transformation frequencies using

different proton doses (5–600 mGy) [3]. They showed

increase in transformation efficiency at doses of 100 and

300 mGy and significant neoplastic transformation fre-

quency was observed at 600 mGy proton radiation dose

[3]. Based on these recent findings, it is clear that a

molecular understanding of proton radiation role in cancer

initiation is vital for space exploration missions and safe

radiation therapies for cancer treatment.

IR can induce mRNA transcriptional changes and

aberrant expression of a variety of non-coding small RNA

in cells. Among these non-coding small RNAs, microRNA

(miRNA) plays a supremely important role in gene regu-

lation through the repression of target genes. Approxi-

mately, 60 % of human protein-coding genes are predicted

to contain miRNA binding sites within un-translated

regions (UTRs) [4]. It has been shown in some studies that

the levels of miRNAs change in response to IRs [5] and

also that miRNAs play an important role in cellular defense

[6, 7].

Several miRNAs have been previously reported as

important players in cancer biology as well as in IR-

induced changes in mouse and human cell lines. In litera-

ture, the up-regulation of miR-100 in mouse plasma after

whole-body c-irradiation (0.5, 2, and 10 Gy), in human

malignant glioma cells and in psoriasis, an autoimmune

disease has been reported [8–10]. In another study, up-

regulation of miR-100 has been shown to down-regulate the

expression of ataxia telangiectasia-mutated (ATM) in

human malignant glioma cells (M059J), which in turn

causes an increased sensitivity to IR [9]. It should also be

noted that the down-regulation of miR-100 was found in

human prostate cancer and normal fibroblast cell lines

exposed to IR/X-rays as compared to the up-regulation in

response to proton radiation [1, 11]. In human tumor

pathology, miR-100 was found to be up-regulated in acute

myeloblastic leukemia (AML) [12] and down-regulated in

some cancers like low-grade bladder cancer, oral cancer,

ovarian cancer, and hepatocellular carcinomas [13–16].

Another miRNA, miR-205, has been previously observed

to be up-regulated in cervical cancer tissues, head and neck

cancer cell lines, squamous cell carcinoma cell lines, and

non-small cell lung cancers [17–23] and down-regulated in

prostate cancer [24, 25]. Several studies have revealed

heterogeneity of miR-205 expression among different

breast cancer subtypes [26–30].

Two independent studies showed the over-expression of

let-7d in normal human fibroblast cell lines exposed to

X-ray (200 cGy/min) treatment, in human dermal micro-

vascular endothelial cells (HDMEC) irradiated with 2 Gy

photon radiation and in c-radiation-treated human thyroid

cells [1, 31, 32]. In another study, irradiation of normal

human lung epithelial line (CRL2741) along with three

lung cancer cell lines (A549, A427 (carcinoma), and

H4441 (adenocarcinoma)) revealed a down-regulation of

let-7 [33]. Three microarray-based cancer experiments

using human tissue also revealed let-7d up-regulation

in lung cancer and down-regulation in pancreatic cancer

[34–37]. In human cancer, miR-423-3p over-expression has

been found in head and neck squamous cell carcinoma

(HNSCC) patients [38], metastatic breast cancer patients

[39], in metastatic neuroblastoma (NB), and hepatocellular

carcinoma tissue (HCC) [40, 41].

Recently, two independent studies reported the down-

regulation of miR-451 in human non-small cell lung car-

cinoma (NSCLC) and gastric and colorectal cancer [42–44]

and significant over-expression of miR-451 among non-

relapsed and relapsed patients with HNSCCs [38].

MiR-146b has been previously found to be up-regulated

in adult papillary thyroid carcinomas (PTCs) with extra-

thyroidal invasion and BRAF mutation and in c-radiation-

treated human thyroid cells [45–49]. While down-regula-

tion of miR-146b in mouse plasma was reported following

whole-body c-radiation [8]. Furthermore, microarray and

qPCR analysis revealed a higher expression of miR-146b in

a murine model of acute and chronic asthma [50].

Recently, Wang and co-workers [42] reported a signif-

icant role of miR-138 on IR-induced DNA damage pathway

by targeting histone H2AX in a human osteosarcoma cell

line [51]. The miR-138 up-regulation and down-regulation

revealed a correlation with chromosomal instability and

cellular sensitivity to DNA-damaging agents [51]. The

down-regulation of miR-138 was also observed in ana-

plastic thyroid carcinoma (ATC) as compared to PTC cell

lines, in which the down-regulation of miR-138 has also

been associated with enhanced telomerase reverse trans-

criptase (TERT) expression [52]. Similarly, reduced miR-

138 level is also associated with enhanced cell growth and

invasion in HNSCC [53, 54].

Furthermore, up-regulation of miR-99b was also repor-

ted in mouse plasma after 2 Gy c-radiation [8]. In human

226 Mol Cell Biochem (2013) 382:225–235

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tumor pathology, miR-99b was up-regulated in patients

with synovial sarcoma [55] and in older adults with

primary melanoma [56]. miRNA expression profiling

revealed a down-regulation of miR-409-5p in human brain

tumor [57], while in another recent study, Nikiforova

reported c-irradiated (1.0 or 10 Gy) human thyroid cells

revealing a high expression of miR-409-5p [32]. Up-regu-

lation of miR-409-5p was also observed in glioblastomas

(GBM) as compared to oligodendrogliomas (ODG) [57],

while a down-regulation of miR-409-5p was observed for

mouse model of Rett syndrome, an X-linked neurological

inherited disorder [58].

Despite the growing evidence of the differential expres-

sion of miRNAs in response to IR [5, 6, 59], little is known

about miRNA response to proton radiation in humans and

mice. Therefore, we sought to examine the effect of proton

irradiation on whole mice with respect to the possible

involvement of miRNA in the onset of cell tumorgenesis. To

this end, we analyzed the global miRNA expression profile in

the brain, liver, and radiation-sensitive testis tissue of proton-

irradiated mice. Our results show that proton radiation can

dysregulated miRNA levels in these three tissues and reveal a

potential pathway by which proton irradiation could induce

pathogenic mutations in miRNA that could lead to cancer.

While further studies are required to define a comprehensive

understanding of tissue-specific miRNA expression profiles

in response to proton radiation, these results show promise

for the possibility that miRNA expression profiles may one

day be useful as diagnostic markers.

Results

MicroRNA expression profiling

In this study, we employed a mouse model to analyze and

compare differential expression of miRNA in brain, liver,

and testis tissues induced by high energy, whole-body

radiation exposure. Eight mice were divided in two groups,

a non-irradiated ‘control’ (group 1) and an irradiated

‘exposed’ group (group 2). Four biological replicates were

used for the control four were used for exposed samples for

brain and testis tissues. These tissues were chosen because

they represent a highly protected tissue (brain) and a highly

sensitive tissue (testis). In the course of study, we recog-

nized that liver tissues would provide a possible interme-

diate exposure and began to collect those data as well. For

liver tissue, therefore, only two biological replicates are

presented for each of the control and exposed samples. The

Illumina HiSeq was used to generate small RNA reads

(2 9 50 bp) isolated from brain, liver, and testis of proton-

irradiated and non-irradiated mice for comprehensive

miRNA differential expression analysis.

Approximately, 87.47, 43.65, and 81.17 million small

RNA reads were obtained for brain, liver, and testis,

respectively (Fig. 1). Out of these total mapped small RNA

reads, 14.71 million (16.79 %) brain miRNA, 1.90 million

(4.59 %) liver miRNA, and 2.71 million (3.27 %) testis

miRNA were obtained after alignment against known

mouse miRNA with 0 mismatches (miRBase revision 17,

http://www.mirbase.org/). We observed a high proportion

(13.67 million; 8.99 %) of testis small RNA reads mapped

to Piwi-interacting RNA (piRNA). A surprisingly large

fraction of reads (28 million; 4.47 % in brain; 4.43 % in

liver and 4.32 % in testes) did not map to any known small

RNA moiety in brain, liver, and testis tissues (Fig. 1).

DESeq software was used for the differential expression

analysis of miRNA data obtained from high-throughput

sequencing. For analysis, strict parameters were used

including higher ‘‘fold change’’ and a p value lower than

0.05. Analysis revealed 30 differentially expressed miRNA

in three mouse tissues (Table 1; Fig. 2).

Mouse brain microRNA differential expression

Cluster analysis revealed a significant dysregulation of eight

miRNAs in proton-irradiated mouse brain tissue compared to

non-irradiated brain tissue (Fig. 2a). The miRNA expression

data showed a good correlation among the four controls and

among the four treated brain biological replicates (0.95 for

0 Gy and 0.95 for 2 Gy). Among these differentially expres-

sed miRNA, one down-regulated (miR3076-3p) and seven up-

regulated (miR-409-5p, miR-205, miR-100, miR-501-3p, miR-

99b, miR-674* and miR-412-5p) miRNAs were observed

(Table 1; Fig. 2a).

Mouse testis microRNA differential expression

We also identified 14 testis miRNAs from the DESeq

analysis that we considered to be differentially expressed

by irradiation at a dose of 2 Gy (Fig. 2b). The miRNA

expression data showed a good correlation among the four

controls and also among the four treated biological repli-

cates (0.90 for 0 Gy and 0.89 for 2 Gy). Among these

differentially expressed miRNA, seven up-regulated (miR-

409-5p, miR-146b, miR-501-3p, miR-99b, let-7d*, miR-

423-3p and miR-720) and seven down-regulated (miR-

1247*, miR-712, miR-467d*, miR-451, miR-138, miR-872*

and miR-497) miRNAs were found (Table 1; Fig. 2b).

Mouse liver microRNA differential expression

Bioinformatics analysis also revealed differential expres-

sion of eight miRNAs among treated and control liver

tissue (Fig. 2c). The miRNA expression data showed a

Mol Cell Biochem (2013) 382:225–235 227

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good correlation between the two controls and also the two

proton-treated biological replicates (0.88 for 0 Gy and 0.86

for 2 Gy). Among these differentially expressed miRNA,

two down-regulated (miR-3473b and miR-10b) and six

up-regulated (miR-3470b, miR-3470a, miR-690, miR-150,

miR-374 and miR-127) miRNAs were observed (Table 1;

Fig. 2c).

Mouse brain, testis, and liver microRNA editing

We performed alignments of mature miRNA reads with

one mismatch to find out miRNA editing patterns in mouse

control (0 Gy) and proton-treated (2 Gy) brain, testis, and

liver tissues. We used only high quality reads and align-

ment of at least 10 reads to remove false positive miRNA

editing events. Approximately, 5.11 % of total reads hav-

ing a single mismatch in mature miRNA reads were

mapped (Fig. 3). We observed a high frequency of single

mismatch reads in brain (*5.61 %) and testis (*4.2 %)

tissues and slightly lower frequency of mismatch in liver

tissues (*2.5 %). We analyzed the frequencies of all

possible single-base substitutions, with particular focus on

the cytosine deamination (C?U (C?T) conversion) and

adenosine deamination (A?I (A?G) conversion).

We observed 24.7, 13.87, and 7.6 % single mismatch

frequencies for T?A, C?T, and A?G substitutions in

brain mature miRNA. We also identified a dominant ratio

(*45.5 %) for A?G single-base substitution in the seed

region of mature brain miRNA (Fig. 3a, b). Liver mature

miRNA reads revealed a higher frequency of C?T

(18.81 %) and A?G (5.22 %) in all substitutions. The

single-base pair mismatch analysis of the seed region

revealed a higher frequency for A?G (12.5 %) as com-

pared to C?T (8.0 %) (Fig. 3a, b). The single mismatch

alignment of testis miRNA reads revealed a higher fraction

of different substitutions. We observed over-representation

of T?A, T?C, A?G, and C?T substitutions, which

accounted for 24.70, 13.92, 12.02, and 8.78 % of single

Fig. 1 Pie charts illustrating the frequency of different non-coding

RNA species. a Pie chart representing brain non-coding RNA

frequencies. Small RNA sequencing revealed a high frequency

(19.86 and 16.79 %) for small nucleolar RNA (snoRNA) and

microRNA (miRNA), respectively, in brain tissue. b Pie chart

representing non-coding RNA frequencies for testes. Small RNA

sequencing revealed a high frequency (21.70 and 8.99 %) for

snoRNA and Piwi-interacting RNA (piRNA), respectively. c Pie

chart showing the liver non-coding RNA frequencies. Small RNA

sequencing revealed a high frequency (20.73 and 5.40 %) for

snoRNA and transfer RNA (tRNA), respectively. Small non-messen-

ger RNA (snmRNA); small cajal body specific RNA (scaRNA); small

nuclear RNA (snRNA); robosomal RNA (rRNA)

228 Mol Cell Biochem (2013) 382:225–235

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mismatch reads, respectively (Fig. 3a, b). We did not

observe any significant differences in miRNA editing fre-

quencies among control (0 Gy) and proton-irradiated

(2 Gy) mouse brain, liver, and testis tissues (Fig. 3a, b).

Discussion

Mouse whole-body proton irradiation (2 Gy) revealed a

significant dysregulation of miRNA in the three tissues

examined (brain, liver, and testis). To approximate the

maximum dose that astronauts may receive from Solar

Particle Events during space missions [60, 61], we used a

2 Gy dose of proton radiation that is equivalent to this

exposure. The tissues used were selected on the basis of

their sensitivity to IR, with brain being the least sensitive,

liver as moderate, and testis as the most sensitive. This

study of selected tissues provides a glimpse of tissue

specificity and sensitivity, as well as several newly iden-

tified, miRNAs whose expression levels change in response

to proton radiation exposure.

Brain differentially expressed miRNA

Mouse brain tissue exposed to 2 Gy proton radiation

treatment revealed a significant up-regulation of seven

miRNAs and down-regulation of one miRNA. Interest-

ingly, we observed an entirely new miRNA expression

profile in proton-irradiated mice brain tissue, which dif-

fered from that observed by Koturbash and co-workers

[62]. This phenomenon suggests a unique miRNA signa-

ture, exclusive to brain tissue that is expressed only in

response to X-ray and proton radiation exposure. The dif-

ference among differentially expressed miRNA can be

attributed to both the radiation dose (1 Gy by Koturbash vs

2 Gy in our study) and the platform used for analysis

(microarray vs next generation sequencing).

Overall, we identified eight dysregulated miRNAs in

proton-irradiated mouse brain and out of these 8, 4 miR-

NAs (miR-99b, miR-100, miR-501-3p and miR-409-5p)

have previously been shown to be dysregulated after

exposure to IR in mice and various human cell lines (see

‘‘Introduction’’ section). The four novel miRNAs whose

expression changed in response to proton irradiation in

mice deserve further study to determine their roles in

IR-induced DNA damage, which could lead to a better

understanding of cancer pathogenesis and ultimately to

earlier diagnosis or new treatments.

Testis differentially expressed miRNA

Small RNA sequencing of testis tissue revealed significant

dysregulation of 14 miRNA (7 up-regulated and 7 down-

regulated). Perplexingly, we did not find any overlap

between these differentially expressed miRNAs and those

identified in a previous microarray study of differential

expression of miRNA in X-ray irradiated mouse testis [63].

The differences observed among differentially expressed

miRNAs may be attributable to the type (X-ray vs proton)

and dose of radiation (2.5 vs 2 Gy) as well as possible

skewing by the analysis platform used (microarray vs next

generation sequencing).

Table 1 MicroRNA species differentially expressed in mouse brain,

liver, and testis after exposure to proton radiation (2 Gy)

MicroRNA Fold change p value

Brain

mmu-miR-409-5pa 4.842 0.000

mmu-miR-205 3.072 0.021

mmu-miR-100 2.530 0.013

mmu-miR-501-3pa 2.380 0.010

mmu-miR-99ba 2.270 0.003

mmu-miR-674b 1.845 0.028

mmu-miR-412-5p 1.546 0.046

mmu-miR-3076-3p 0.064 0.009

Testis

mmu-miR-409-5pa 3.796 0.014

mmu-miR-146b 2.668 0.042

mmu-miR-501-3pa 2.360 0.017

mmu-miR-99ba 2.186 0.035

mmu-let-7db 2.016 0.001

mmu-miR-423-3p 1.780 0.004

mmu-miR-720 1.548 0.046

mmu-miR-1247b 0.000 0.039

mmu-miR-712 0.282 0.035

mmu-miR-467db 0.475 0.023

mmu-miR-451 0.514 0.002

mmu-miR-138 0.543 0.005

mmu-miR-872b 0.600 0.023

mmu-miR-497 0.606 0.017

Liver

mmu-miR-3470b 5.857 0.000

mmu-miR-3470a 5.115 0.001

mmu-miR-690 3.937 0.009

mmu-miR-150 3.014 0.011

mmu-miR-374 2.662 0.050

mmu-miR-127 1.756 0.030

mmu-miR-3473b Inf 0.003

mmu-miR-10b 0.755 0.031

a Indicates the overlapping microRNA expressing among brain and

testis tissuesb Indicates an miRNA expressed at low levels relative to the miRNA

in the opposite arm of a hairpin

Mol Cell Biochem (2013) 382:225–235 229

123

Here, we report a significant up-regulation (2.0-fold

change) of mmu-let-7d* a let-7 family member in response

to proton radiation. These results correlate well with a

variety of previously observed changes in let-7 family

genes upon exposure to radiation [1, 31–37]. The second

most highly expressed miRNA found in exposed testis

tissue was miR-146b (fold change-2.66). In our analysis,

we also observed up-regulation (1.7-fold change) of miR-

423-3p following proton irradiation in mice, which sug-

gests a possible role in cell cycle and growth. Together

with previous reports, these findings suggest that miR-423-

3p might function as an oncogenic miRNA in HCC and

other cancers [38–41].

In this study, we identified that miR-451 showed low

expression (fold change 0.51) in proton-treated mouse testis,

consistent with reports of its dysregulation in various cancers

[38, 41, 44]. In our hands, down-regulation (0.54-fold change)

of miR-138 was found in proton-irradiated mouse testis. As

described above, extensive work has implicated that radiation-

induced damage to miR-138 expression correlates with DNA

damage, damage pathways, and pathogenic conditions [51].

The down-regulation of miR-497 observed here in proton-

irradiated mouse testis tissue is consistent with a similar

observation in adrenocortical carcinoma (ACC), human

breast cancer tissue and cell lines, colon, prostate, ovarian,

gastric, lung cancers, multidrug-resistant human gastric can-

cer cell line (SGC7901/VCR) and a multidrug-resistant

human lung cancer cell line (A549/CDDP) [64–69].

Other mouse testis miRNAs found to be differentially

expressed on exposure to proton irradiation in this study,

four down-regulated (miR-1247*, miR-712, miR-467d*,

and miR-872*) and one up-regulated (miR-720), have not

been previously reported in association with radiation

exposure and cancer.

Fig. 2 Differentially expressed miRNAs in mouse control (0 Gy) and

proton-irradiated (2 Gy) brain, liver, and testis tissues. MicroRNA

found to be differentially expressed after exposure to 2 Gy of proton

radiation in at least one tissue are represented as rows in this heat

map. Each sample was exposed to 0 or 2 Gy radiation as indicated by

sample labels. The final digit of the sample label refers to the replicate

number. The color of each box represents the expression level of an

miRNA in a given sample, where green represents low expression and

red represents high expression. The expression of each miRNA is

normalized within each tissue type. Orange boxes delineate groups of

miRNAs found to be over expressed within that tissue type while

those in purple boxes were found to be under expressed. Three

miRNAs were found to be over expressed in both brain and testis.

a Samples from brain tissues, labeled MB. b Samples from testis

tissues, labeled MT. c Samples from liver tissues, labeled ML. (Color

figure online)

230 Mol Cell Biochem (2013) 382:225–235

123

Overlapping dysregulated miRNAs in brain and testis

Three same miRNAs were observed to be dysregulated

against proton irradiations in mice brain and testis tissues.

The miR-99b over-expression in mouse brain and testis is

consistent with up-regulation in mouse plasma after 2 Gy

c-radiation [8], in synovial sarcoma patients [55] and in

older adults with primary melanoma [55]. The highest up-

regulation of miRNA (miR-409-5p) was found in both

brain and testis tissues of proton-irradiated mice, in keep-

ing with previous reports of dysregulation of miR-409-5p

in a variety of neurological cancers [32, 57, 58] but with

unknown implications for cancers related to testis tissue.

Over-expression of miR-501-3p (with more than a twofold

change) was observed in proton-irradiated mouse brain and

testis. This is in keeping with recently reported up-regu-

lation of miR-501-3p in mouse plasma after whole-body

2 Gy c-irradiation [8].

Liver differentially expressed miRNA

Small RNA sequencing of proton-irradiated mouse liver tis-

sue revealed significant dysregulation of eight miRNAs (6 up-

regulated and 2 down-regulated). MiR-150, a hematopoietic

specific miRNA, important in B and T lymphocytes differ-

entiation showed up-regulation in proton-irradiated mouse

liver, a finding that is consistent with similar observations in

gastric cancer, hepatoblastoma, osteosarcoma samples and

non-small cell lung cancer [70–74]. Down-regulation of miR-

150 was found for Natural Killer/T-cell lymphoma, malignant

pancreatic tissues and small cell lung cancer [74–76].

MiR-127, a putative tumor suppressor, was also found to

be up-regulated (fold change 1.75) in proton-irradiated

mouse liver. By contrast, Wagner-Ecker et al. [31] found

down-regulation of miR-127 in response to 2 Gy photon

radiation in HDMEC [31]. In human tumor pathology,

miR-127 was previously found to be up-regulated in lymph

node metastasis [77], Epstein-Barr virus EBV-positive

Burkitt lymphoma (BL) [78] and down-regulated in a

methyl-deficient rat model of liver carcinogenesis [79]. We

observed higher expression of miR-374 (2.66-fold change)

in proton-irradiated mouse liver, which is consistent with

previously observed up-regulation in three small cell lung

cancer cell lines (HTB-184, HTB-172, and H69) [74].

In this study, down-regulation (0.75-fold change) of

miR-10b was found in proton-irradiated mouse liver, which

is consistent with down-regulation of miR-10b in breast

cancer and in genetically engineered mouse (GEM) models

of human breast cancer [27, 80]. Similarly, Ma et al. [81]

reported a down-regulation of miR-10b in human and

mouse primary breast cancer and higher expression in

metastasis breast cancer [81].

Out of the eight miRNAs that we observed to be

dysregulated in proton-irradiated mouse liver, one down-

regulated miRNA (miR-3473b) and three up-regulated

miRNAs (miR-3470b, miR-3470a, and miR-690) have not

Fig. 3 MicroRNA editing pattern in control (0 Gy) and proton-irradiated (2 Gy) mouse brain, liver, and testis tissues. a Single-base pair

substitutions or miRNA editing frequencies in full-length mature miRNAs b miRNA editing frequencies in mature miRNA seed regions

Mol Cell Biochem (2013) 382:225–235 231

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been previously reported in association with radiation

exposure and cancer.

Editing of brain, liver, and testis miRNA

Post-transcriptional modification or editing of mammalian

primary-miRNAs (pri-miRNA) has a large impact on

processing and functionality of mature miRNAs and thus

affects expression and regulation of target genes [82, 83].

Efficient target mRNAs regulation requires a perfect base-

pairing of seed region (2–8 nucleotides) from the 50-end of

mature miRNA [84]. The editing events within the highly

conserved seed sequence can increase and decreases the

specificity as well as the numbers of target genes [82, 83,

85]. In this study, mature miRNA showed higher adenosine

deamination (A?I) frequency in mouse brain. The higher

adenosine deamination phenomenon can be attributed to

the brain-specific expression of the mammalian adenosine

deaminases (ADARs) responsible for A?I editing. We

observed a *6.0 % higher A?G substitution frequency in

the seed region than the full-length mature miRNA

(Fig. 3a, b). In contrast, the frequency of C?T substitu-

tions in the seed region was *3.0 % lower when compared

to mature miRNA (Fig. 3a, b). Similarly to brain miRNA,

we observed a *2.5 % increase in the A?G substitution

ratio and a *2.5 % decline in the C?T substitution ratio

for the seed region as compared to mature miRNA in liver

tissue (Fig. 3a, b). Previously, Joyce et al. [10] demon-

strated through deep sequencing of human psoriasis skin

that a higher frequency of adenosine deamination and a

lower frequency of cytosine deamination occurred in the

seed region as compared to mature miRNA [10]. In testis

miRNA reads, we observed a lower frequency of A?G

and C?T substitutions in mature miRNA sequence as well

as in the seed region. Meanwhile, we observed higher

frequencies (24.70 and 16.5 %) for T?A and T?C sub-

stitutions in full-length mature miRNA as well as the seed

region (Fig. 3a, b). The functional significance of miRNAs

A?I editing catalyzed by adenosine deaminases has been

proven in various research [83, 86]. Tissue specific A?I

editing within the seed region of mature hsa-miR-376

cluster transcript can lead to modifications in the target

recognition of mRNA targets in some tissues [83].

Conclusion

Deep sequencing of small RNA species revealed a unique

miRNA expression signature and post-transcriptional

modification for each tissue (brain, lever, and testis) from

whole-body proton-irradiated mice. To date, there have

been limited studies showing the response of these three

cell-types on miRNA expression following low doses of

proton radiation. All three tissues assessed in this study

showed differentially expressed profiles, with testis, the

most sensitive tissue, showing the greatest number of

responding miRNAs. Overall, this study has identified

several miRNAs that may be associated with tumorgenesis

in direct response from proton radiation. Further validation

and characterization studies on these altered miRNAs

could be helpful in elucidating the cellular and genetic

changes induced by proton irradiation, and in understand-

ing possible links between proton irradiation and cancer.

Materials and methods

Samples and tissues

Balb/C male mice were purchased from Harlan Laboratories

Inc. (Livermore, USA). The mice were irradiated with

protons after proper resting for 2 days at the Loma Linda

University Radiation Facility (Loma Linda, CA). Mice in

Group 1 served as a control (0 Gy), while mice in Group 2

were exposed (2 Gy) to a proton source at a dose rate of

1Gev/45 s. The controls and irradiated mice were eutha-

nized 4 h after exposure; brain, liver, and testis tissues were

then dissected out and immediately frozen in liquid nitrogen.

The animal care and treatment procedures were reviewed

and approved by the Institutional Animal Care and Use

Committees (IACUC) at the Loma Linda radiation facility.

RNA isolation and small RNA library preparation

for microRNAs

Total RNA was extracted from tissues (brain, liver, and

testis) using Trizol reagent (Invitrogen; Carlsbad, CA) per

manufacturer’s instructions. After passing quality controls

(quantification and quality check by Agilent 2100 Bioan-

alyzer), 1 lg of total RNA, without selection of small

RNAs, was used directly in Illumina TruSeq Small RNA

Sample Prep Kits (Catalog # RS-200-0012) to prepare

libraries for the three different tissues as well as controls.

Four biological replicates were used for brain and testis

tissue, while due to availability issue, only two biological

replicate were used for liver tissue. The quality of libraries

was determined on the Agilent 2100 Bioanalyzer (Agilent;

Palo Alto, CA) per manufacturer’s recommendation.

MicroRNAs sequencing

The bar-coded small RNA libraries for each irradiated and

non-irradiated mouse tissue (brain, liver, and testis)

including biological replicates were pooled together in

equal concentrations in one pool for sequencing analysis.

The pooled libraries were subjected to pair-end (2 9 50

232 Mol Cell Biochem (2013) 382:225–235

123

bp) sequencing using two different lanes on the HiSeq

2000 (Illumina, Inc) to analyze differential miRNAs

expression patterns.

Mapping and differential expression of microRNAs

sequencing data

The miRNA expression data was generated from two

Illumina HiSeq runs merged together to account for the

lack of counts identified from a single HiSeq run. For each

run, the paired end reads were first merged using SeqPrep

(https://github.com/jstjohn/SeqPrep) and then cleaned up

by removing any TruSeq adapters identified. After the

reads were merged, the data was filtered such that only the

size range for known mouse miRNAs from miRBase

revision 17 [87] remained. Bowtie [88] with miRBase v17

was run with no mismatches to identify mature miRNA

counts from the Illumina HiSeq data. Counts from both of

the runs were added together. Statistically significant

miRNAs for each tissue were identified using DESeq [89]

development version (R-devel build 09/01/2011) by

selecting for an unadjusted p value \0.05.

MicroRNA editing analysis

Possible RNA editing events were identified by looking for

substituted bases in Bowtie aligned reads. Mature miRNAs

from miRBase (revision 17, http://www.mirbase.org) were

used as a reference and a single substitution was allowed

during alignment. Observed substitution rates were gener-

ated separately for control (0 Gy) and proton-irradiated

(2 Gy) tissues by dividing the number reads aligning to

miRNAs which contained one substitution by the total

number of aligned reads (i.e. reads containing zero or one

substitutions).

Acknowledgments This study was supported in part by grants from

the National Aeronautics and Space Administration Cooperative

Agreements NCC9-165 and NNX08BA47A, National Institutes of

Health [P01-HG000205], the National Science Foundation [DBI

0830141].

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