Life Sciences in Space Research 1 (2014) 44–52

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Life Sciences in Space Research

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Changes in miRNA expression profile of space-flown Caenorhabditiselegans during Shenzhou-8 mission

Dan Xu, Ying Gao, Lei Huang, Yeqing Sun ∗

Institute of Environmental Systems Biology, Dalian Maritime University, Linghai Road 1, Dalian, 116026, PR China

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 October 2013Revised 20 November 2013Accepted 25 November 2013

Keywords:C. elegansSpaceflightMicroarrayDauer larvaemiRNATarget genes

Recent advances in the field of molecular biology have demonstrated that small non-coding microRNAs(miRNAs) have a broad effect on gene expression networks and play a key role in biological responses toenvironmental stressors. However, little is known about how space radiation exposure and altered gravityaffect miRNA expression. The “International Space Biological Experiments” project was carried out inNovember 2011 by an international collaboration between China and Germany during the Shenzhou-8(SZ-8) mission. To study the effects of spaceflight on Caenorhabditis elegans (C. elegans), we exploredthe expression profile miRNA changes in space-flown C. elegans. Dauer C. elegans larvae were takenby SZ-8 spacecraft and experienced the 16.5-day shuttle spaceflight. We performed miRNA microarrayanalysis, and the results showed that 23 miRNAs were altered in a complex space environment anddifferent expression patterns were observed in the space synthetic and radiation environments. Mostputative target genes of the altered miRNAs in the space synthetic environment were predicted to beinvolved in developmental processes instead of in the regulation of transcription, and the enrichmentof these genes was due to space radiation. Furthermore, integration analysis of the miRNA and mRNAexpression profiles confirmed that twelve genes were differently regulated by seven miRNAs. These genesmay be involved in embryonic development, reproduction, transcription factor activity, oviposition in aspace synthetic environment, positive regulation of growth and body morphogenesis in a space radiationenvironment. Specifically, we found that cel-miR-52, -55, and -56 of the miR-51 family were sensitive tospace environmental stressors and could regulate biological behavioural responses and neprilysin activitythrough the different isoforms of T01C4.1 and F18A12.8. These findings suggest that C. elegans respondedto spaceflight by altering the expression of miRNAs and some target genes that function in diverseregulatory pathways.

© 2014 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.

1. Introduction

The space environment is complex and is characterised by highlinear energy transfer (LET) radiation, ultra-high vacuum, a weakmagnetic field, microgravity and other phenomena (Kiefer andPross, 1999; Reitz, 2008). Among these physical mechanisms, spaceradiation and microgravity are the most important. Space radiation,which contains high-energy particles that can cause fundamentalcellular changes in human tissues and possibly increase chromo-somal aberrations and cancer risks, significantly differs from thetypes of radiation on earth (George et al., 2007; Maalouf et al.,2011). Microgravity (10−4–10−6 g) in space differs from gravity(1 g) on earth. Microgravity can cause visual disorders, muscle al-

* Corresponding author. Tel.: +86 411 84723633 888; fax: +86 411 84725675.E-mail addresses: jotan1995@163.com (D. Xu), gaoying8000@126.com (Y. Gao),

haihai7374@163.com (L. Huang), yqsun@dlmu.edu.cn (Y. Sun).

http://dx.doi.org/10.1016/j.lssr.2013.12.0012214-5524/© 2014 The Committee on Space Research (COSPAR). Published by Elsevier L

terations, bone loss and the dysfunction of the cardiovascular sys-tem (Crawford-Young, 2006; Narici et al., 2004). Therefore, spacebiological research efforts have focused on whether space micro-gravity has a synergistic effect on space radiation-induced damageresponse.

In the past, animals such as Caenorhabditis elegans (C. elegans),mice and rats have been used as model systems to support hu-man exploration of space and understand the biological changesin humans during spaceflight (Morey-Holton et al., 2007). Studiesbased on these model organisms have shown that space radia-tion and microgravity can induce DNA damage, mutagenesis, andgenomic instability (George et al., 2007; Kiefer and Pross, 1999;Zhao et al., 2006). C. elegans, a small roundworm (nematode),is a suitable model organism for accessing and monitoring con-ditions in space. These worms are characterised by short bodylengths, distinct life cycle processes and a strong stress resistancecapacity. They have a well-understood genome background, with40% homology to the human genome (Hu, 2007). It is possible

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D. Xu et al. / Life Sciences in Space Research 1 (2014) 44–52 45

to study certain biological effects using the proper mutants in awell-understood genetic background. In the past several decades,C. elegans has been flown on a number of shuttle missions and hasalso been on the International Space Station (Adenle et al., 2009;Hartman et al., 2001; Jamal et al., 2010). Studies from the In-ternational C. elegans Experiment First Flight (ICE-First) projectdemonstrated the biological effects of short duration spaceflight(11 days on the International Space Station) on individual de-velopment, reproduction, apoptosis, gene stability and the muta-tion rate (Adenle et al., 2009; Szewczyk et al., 2008). Many re-search groups have also contributed to the understanding of thesebiological changes in a complex space environment using othermodel organisms (Adachi et al., 2008; Higashibata et al., 2006;Selch et al., 2008). However, the genetic and epigenetic regulatorymechanisms that are active in space remain to be elucidated.

It is known that microRNAs (miRNAs) are a class of non-codingRNA that control gene expression through translational inhibi-tion and the destabilisation of messenger RNA (mRNA) targets atthe post-transcriptional level (Davis and Hata, 2009; Holtz andPasquinelli, 2009). The first two miRNAs identified, lin-4 and let-7,were originally found in C. elegans (Lee et al., 1993; Reinhart etal., 2000). Subsequently, more than 200 miRNAs have been dis-covered in C. elegans, and these miRNAs have been implicated indevelopmental timing, cell differentiation, life span and apoptosis(Kloosterman and Plasterk, 2006; Stefani and Slack, 2008). As en-dogenous non-coding small RNAs, miRNAs are sensitive to environ-mental stress, and altering the expression of miRNAs is an impor-tant mechanism by which cells adjust to different environments.Previous work has shown that miRNA expression profiles are al-tered by exposure to simulated microgravity or radiation, such asX-ray, ultraviolet (UV) and photon irradiation (Khan et al., 2013;Mangala et al., 2011; Zhou et al., 2012). Several miRNAs, includ-ing miR-150, miR-34a, miR-423-5p, miR-22, miR-141, miR-618,and miR-222, were significantly up-regulated in TK6 human lym-phoblastoid cells in the simulated microgravity condition (Mangalaet al., 2011). Modelled microgravity altered the miRNA expres-sion signature of irradiated human lymphocytes by decreasing thenumber of radio-responsive miRNAs, which can affect the DNA-damage response to ionising radiation through the miRNA-mRNAinteraction mechanism (Girardi et al., 2011). We propose that theanalysis of the changes in miRNA and target gene expression willbring to light the effects of complex space factors such as micro-gravity and space radiation on the biological response of C. elegansflown in space.

In 2011, China successfully launched Shenzhou-8 (SZ-8) viaa Long March 2F launch vehicle at the southern launch site ofthe Jiuquan Satellite Launch Center. There were a total of 17research programs in the “International Space Biological Experi-ments” project on board SZ-8, including Project SIMBOX, a coop-erative project between China and Germany. The samples utilisedfor the space experiments included plants, animals and humancells that were exposed to the space environment for a period ofthree weeks. Among these samples, dauer C. elegans larvae wereselected as a model organism to avoid interference between thedifferent generations and the different developmental stages. Thedauer stage is an alternative developmental pathway for C. ele-gans worms that are exposed to crowded or low food conditions.Dauer larvae can survive in adverse conditions for several monthswithout feeding. Genetic characteristics can be inherited by theoffspring after breeding (Hu, 2007; Onodera et al., 2010). Dauer. elegans larvae are considered an appropriate model system to de-velop future biomedical research. In the future, they might act asbiological sensors that access the effects of space radiation and mi-crogravity during both near-term short duration and future longermissions. In our study, the dauer larvae were confirmed to be alive

after the end of the SZ-8 mission and could, therefore, be used forfurther analysis of the biological effects of space.

We performed our experiments on SZ-8 to study the biolog-ical effects of space radiation and microgravity with the aid ofthe SIMBOX device. SIMBOX is the first non-Chinese experimentalequipment and is used as a China-German cooperative universalbiological culture device. According to the International Space Sta-tion experimental standards, the device was designed to providetemperature, light and other life-support conditions. We could es-tablish two spaceflight groups in the SIMBOX device: 1) samplesthat were in a fixed state, suffering space radiation and micro-gravity; and 2) samples that were in a rotating system that wasused to imitate Earth’s gravity (1 g). This system used the cen-trifugal state to negate the effects of microgravity; therefore, thesesamples could be used as space controls to independently studyspace radiation. Thus, a comparison between the two groups couldreflect the influence of microgravity on the biological effects ob-served during space missions.

It has been reported that the changes in miRNA expression ap-pear 4 h after irradiation, and many more miRNAs are alteredat 24 h after irradiation (Girardi et al., 2012). Thus, studying themiRNA expression profile within 24 h after environmental stimulicould provide a better understanding of the biological responsesto the space environment. In our study, C. elegans samples werecollected and fixed 7 h after the SZ-8 spacecraft returned. Com-plete RNA samples were isolated from approximately 2000 wormsfrom each group for miRNA and mRNA expression analysis. Weanalysed the miRNA expression profiles of space-flown C. elegans,and the results showed different expression patterns of miRNAsbetween the space radiation and space synthetic environments.Prediction and integration of the target genes demonstrated thata number of the altered miRNAs function broadly through the reg-ulation of multiple anti-corrected targets, most likely due to theirinvolvement in embryonic development, reproduction, locomotion,behaviour and other biological responses to spaceflight.

2. Methods

2.1. Culture conditions and synchronisation process of C. elegans

A Bristol N2 (wild-type) strain of C. elegans was obtainedfrom the Caenorhabditis Genetics Center (Minneapolis, MN, USA).The worms were cultured at 23 °C on nematode growth medium(NGM) agar with E. coli OP50 as a food source. The worms en-tered the dauer stage through a synchronisation process prior tothe spaceflight experiments. Briefly, adult worms were collectedand soaked in a worm lysis solution (0.5 ml of 5 N NaOH, 0.5 ml of10% household bleach, 3 ml of sterile H2O) for 10 min. The wormswere centrifuged at 1300 g for 30 s to isolate the embryos andwashed with sterile H2O two to three times. Then, the embryoswere transferred to fresh standard NGM plates where the contin-uously developing larvae were prepared. Dauer larvae were thenselected by washing with 1% SDS and harvested immediately, asthe dauer stage is defined by SDS resistance.

2.2. Spaceflight experiment

Dauer C. elegans larvae were divided into three groups (Table 1):(1) spaceflight samples (SF) were put in a fixed device, and theseworms were affected by space synthetic factors, mainly space ra-diation and microgravity; (2) spaceflight control samples (SC) wereput in a centrifugal device, and these worms were affected byspace radiation, but not microgravity as the rotating system wasdesigned to imitate Earth’s gravity (1 g); (3) ground control sam-ples (GC) were cultured under Earth’s gravity (1 g) and were usedas a control.

46 D. Xu et al. / Life Sciences in Space Research 1 (2014) 44–52

Table 1Experimental groups and indicated meaning.

# Groups Symbol Gravity Radiation Microgravity Indicated meaning

1 Spaceflight SF μ g + + Space synthetic factors2 Spaceflight SC 1 g + – Space radiation3 Ground control GC 1 g – – Used as control

Fig. 1. Experimental culture containers and equipments on broad SZ-8. Dauer larvae of C. elegans were put in the two culture containers (A), and the containers weretransferred to SIMBOX device (B) during SZ-8 mission. The samples can be fixed on the outer ring of SIMBOX device in fixed state, and they can be also put in the inner ringof SIMBOX device in centrifugal state. Both fixed state and centrifugal state are indicated by arrows.

For the spaceflight worms, approximately 105 dauer wormswere put into two experimental culture containers containing freshNGM (Fig. 1A). The containers were covered with a transparentbiofilm and transferred into the special SIMBOX device (Fig. 1B),which allows for air exchange between the environment and theexperimental culture boxes. The worms experienced the 16.5 daylong shuttle flight aboard SZ-8 from Nov 1 through Nov 17, 2011.During this period, they experienced space radiation (1.92 and2.27 mGy dose measured by the thermoluminescent detector (TLD)in the fixed device and centrifugal device, respectively) and micro-gravity. Seven hours after landing, the worms were carried backand immediately observed under a light microscope. We con-firmed that these C. elegans were alive and had remained in thedauer stage in each group. Then, the samples were fixed with liq-uid nitrogen and maintained until they were returned to DalianMaritime University. In parallel, the worms in the ground controlgroups were cultured on Earth in the Beijing PITC Space Center forthe same 16.5 days and were used as controls for further analysis.

2.3. miRNA and mRNA microarray analysis

Total RNA was extracted from the four groups of C. elegans(approximately 2000 worms) using TRIzol (Invitrogen) and amiRNeasy mini kit (QIAGEN) according to the manufacturers’ in-structions. Total RNA from each sample was quantified using aNanoDrop ND-1000, and RNA integrity was assessed using stan-dard denaturing agarose gel electrophoresis analysis. miRNA mi-croarray analysis and gene expression profiling analysis were per-formed by KangChen Bio-tech Inc. (Shanghai).

For the miRNA expression profiles, the RNA samples were la-belled using the miRCURY™ Hy3™/Hy5™ Power labelling kit andhybridised to the miRCURY™ LNA Array (v.11.0). Following thewashing steps, the slides were scanned using the Axon GenePix4000B microarray scanner (Axon Instruments, Foster City, CA).Scanned images were then imported into the GenePix Pro 6.0software (Axon) for grid alignment and data extraction. Repli-cated miRNAs were averaged, and miRNAs with intensities � 50in all samples were chosen for calculation as normalisation factors.Expressed data were normalised using Median normalisation. Af-ter normalisation, the differentially expressed miRNAs (� 1.5-fold

changes) were identified through fold change filtering. The expres-sion level of each miRNA was calculated as fold change.

For gene expression profiling, approximately 5 μg of the RNAsamples were labelled and hybridised to the array. Gene expres-sion profiling included the following steps: 1) reverse transcriptionusing a Invitrogen Superscript ds-cDNA synthesis kit, 2) ds-cDNAlabelling with a NimbleGen one-colour DNA labelling kit, 3) ar-ray hybridisation using the NimbleGen Hybridisation System, fol-lowed by washing with the NimbleGen wash buffer kit and 4)array scanning using the Axon GenePix 4000B microarray scanner(Molecular Devices Corporation). Scanned images were importedinto the NimbleScan software (version 2.5) for grid alignment andexpression data analysis. All gene level files were imported intothe Agilent GeneSpring GX software (version 11.5.1) for furtheranalysis. Pathway analysis and gene ontology (GO) analysis wereperformed to determine the roles of the differentially expressedgenes (� 2-fold changes).

3. Results

3.1. Altered miRNA expression in response to spaceflight

C. elegans were carried into space, where they experiencedspace radiation and microgravity during the flight. The miRNA ex-pression of C. elegans after spaceflight was measured using a mi-croarray covering 157 miRNAs. Compared with the ground controlgroup, the expression of 23 miRNAs changed when exposed to aspace synthetic environment (R+M) and a space radiation envi-ronment (R), as shown in Table 2. These miRNAs exhibited threedifferent expression patterns. First, miRNAs, including miR-56, 82,84, 124, 237, 256 and 796, were up-regulated or down-regulatedin both the R+M and R conditions, indicating that these miRNAswere affected by the space synthetic environment due to space ra-diation. Second, miRNAs, including miR-52, 81, 257, 265, 795, 1822and 1823, were only occurred in the R+M condition, indicatingthat they might be affected by microgravity in a space syntheticenvironment. Third, miRNAs, including miR-55, 73, 230, 235, 258,787, 788, 799 and 1824, were only changed in the R condition,indicating that they could be affected by space radiation, with anegligible effect from microgravity. These findings suggest that themiRNA expression profile was significantly altered by spaceflight,

D. Xu et al. / Life Sciences in Space Research 1 (2014) 44–52 47

Table 2Differentially expressed miRNAs in spaceflight groups versus ground control of C. el-egans.

miRNA name Space synthetic environment(R+M)

Space radiation(R)

cel-miR-256 −14.34 −21.72cel-miR-124 −2.01 −1.86cel-miR-84 −1.76 −2.64cel-miR-82 −7.87 −5.45cel-miR-796 −2.30 −2.00cel-miR-56 1.79 2.00cel-miR-237 1.81 2.36cel-miR-81 −4.65cel-miR-257 1.74cel-miR-795 2.88cel-miR-265 −4.12cel-miR-1823 −1.74cel-miR-52 1.79cel-miR-1822 2.90cel-miR-799 2.00cel-miR-230 −1.91cel-miR-258 −2.23cel-miR-73 1.85cel-miR-55 1.91cel-miR-788 2.97cel-miR-1824 2.68cel-miR-787 1.55cel-miR-235 24.54

Note: miRNA expression profile was analysed and the expression level of eachmiRNA were presented as fold changes (R+M: SF vs GC; R: SC vs GC).

and the alteration of the miRNA expression levels exhibited a di-versity of expression patterns in different space conditions.

3.2. Target prediction and integration analysis of miRNA target genes

The prediction and integration analysis schematic for themiRNA target genes is shown in Fig. 2. In summary, we isolatedand grouped the dauer larvae into spaceflight and on-the-groundgroups, followed by data analysis of the miRNA and mRNA ex-pression profiles. The analysis focused on the miRNA target genesthat were commonly predicted by Miranda, PicTar and TargetScan-Worm. Finally, we preformed GO analysis and analysed the func-tion correlation between the miRNAs and mRNAs.

3.2.1. Target prediction of miRNA target genesTo predict the target genes of the differentially expressed miR-

NAs, we first performed computational analysis with a combi-nation of Miranda, PicTar and TargetScanWorm. Focusing on thecommonly predicted target genes of the differentially expressedmiRNAs, we classified these genes using DAVID to determine whichGO terms were enriched (Fig. 3).

We analysed the miRNA putative genes in an R environmentand found that “regulation of transcription” was the most enrichedbiological process, followed by “body morphogenesis”, “regulationof RNA metabolic process”, “phosphorylation”, “behaviour”, “ovipo-sition” and “positive regulation of locomotion”. All of these pu-tative genes analysed in the R environment also appeared in theR+M environment, which indicates that they might be affected byspace radiation.

In contrast, biological categories including “embryonic develop-ment”, “post-embryonic development”, “nematode larval develop-ment”, “reproductive development”, “sex differentiation” and “gen-italia development” were more enriched in the R+M environ-ment. Interestingly, the developmental biological processes werenot enriched in the R environment. Additionally, “localisation ofcell”, “cellular protein localisation”, “cellular macromolecule local-isation”, “cell motility” and “cell migration” were only enriched inthe R+M environment, but not in the R environment. These resultssuggest that there are significant differences in the miRNA putativetarget genes between the R+M and R environments, which mightbe attributed to the effects of microgravity in space synthetic con-ditions.

3.2.2. Integration analysis of miRNA target genesBased on computational predictions, we analysed putative

miRNA-mRNA target interactions to identify the most likely targets.Previous work has shown that miRNAs negatively regulate geneexpression at the post-transcriptional level. Therefore, we searchedfor up-regulated miRNAs and the corresponding down-regulatedmRNA targets between putative pairs of miRNA and mRNA in thedifferent conditions. Similarly, down-regulated miRNAs and up-regulated targets were also examined under the different environ-mental conditions, as shown in Table 3. We found that there were12 significant anti-correlated target genes for 7 altered miRNAs

Fig. 2. The procedure of computational prediction and analysis of target genes. Dauer larvae of C. elegans were collected to isolate total RNA, followed by miRNA and mRNAmicroarray analysis. To predict the target genes of differentially expressed miRNAs, computational analysis with combination of Miranda, PicTar and TargetScanWorm wasperformed. Then, those putative gene expression was studied to reveal putative miRNA-mRNA target interaction. Focus on the common predicted target genes of miRNAs,functional correlation between miRNA and mRNA expression profiling was further investigated.

48 D. Xu et al. / Life Sciences in Space Research 1 (2014) 44–52

Fig. 3. Gene ontology (GO) analysis of putative target genes of altered miRNAs. The putative target genes of differentially expressed miRNAs under space synthetic environment(R+M) and space radiation (R) were classified according to DAVID. Biological categories of target genes between the two dashed lines appeared under both R+M and Renvironment, while other genes outside the dashed lines only appeared under R+M environment.

Table 3Anti-correlated genes of differentially expressed miRNAs in spaceflight groups versus ground control of C. elegans.

miRNAs EnsemblGene ID

Genesymbol

GO Terms Space factorsa

R+M R

cel-miR-81,82 F55C5.4 capg-2 embryonic development; locomotion; ↑ –positive regulation of growth

cel-miR-124 ZC477.9 deb-1 reproduction, structural molecule activity ↑ –cel-miR-81 ZK180.5 ZK180.5 transcription factor activity ↑ –cel-miR-56 C08C3.1 egl-5 oviposition, DNA binding ↓cel-miR-52, 56 C07H4.1 C07H4.1 membrane ↓ –cel-miR-84, 124 F41D9.3 wrk-1 Axon guidance ↑ –cel-miR-52,55,56 F18A12.8 nep-11 neprilysin activity ↓ ↓cel-miR-52,55,56 T01C4.2 odr-2 behaviour ↓ ↓cel-miR-82 K11G9.4 egl-46 positive regulation of growth, nucleic acid binding – ↑cel-miR-124 C26D10.5 eff-1 phospholipase A2 activity, post-embryonic body morphogenesis – ↑cel-miR-82 H13N06.2 H13N06.2 unknown ↑ –cel-miR-124 F57B9.7 F57B9.7 unknown ↑ –

Note: a Factors annotates the changes of gene expression are affected by which factors: R+M indicates space radiation and microgravity; R indicates space radiation. Thechanges in genes expression were indicated by up-regulated (↑) or down-regulated (↓) or no changes (–).

(cel-miR-52, 55, 56, 81, 82, 84 and 124) in the space synthetic en-vironment and/or the space radiation environment (Table 3).

Integration analysis of the miRNA and mRNA expression pro-files refined the functional miRNA-mRNA relationship. First, weanalysed the target genes of the altered miRNAs in the R+M en-vironment and found that these genes may be involved in manyaspects of the predicted GO terms. For example, F55C5.4, the com-mon target gene of cel-miR-81 and 82, was up-regulated andhas also been implicated in embryonic development, locomotion,and the positive regulation of growth. Cel-miR-124 could regu-late the corresponding gene ZC477.9, which was up-regulated andmight function in reproduction and structural molecular activity.Cel-miR-81 could target ZK180.5, which affects transcription factor

activity. F41D9.3, a common target of cel-miR-84 and 124, was up-regulated and is possibly involved in axon guidance during neurondevelopment. We also observed the down-regulation of C08C3.1and C07H4.1 in the R+M environment, which corresponds to theup-regulation of cel-miR-52 and miR-56, respectively. These genesmight be involved in oviposition and membrane-related biologi-cal processes. Notably, these target genes were only altered in theR+M environment, not in the R environment. This indicates thatthese genes could play a role in the biological processes affectedby microgravity.

In contrast, we analysed the anti-correlated genes of the al-tered miRNAs in the R environment. The K11G9.4 gene, whichis targeted by miR-82, was predicted to function in the positive

D. Xu et al. / Life Sciences in Space Research 1 (2014) 44–52 49

Fig. 4. Alignment of sequence for cel-miR-51 family. The members of cel-miR-51family are highly conserved. Bases 2–8 (“seed region”) are highlighted by blackbackground. The same other bases among miR-52, miR-55 and miR-56 were in-dicated by grey background. The percentages of sequence similarity (miR-55 vsmiR-52; miR-56 vs miR-52 ) were shown in the right region, respectively.

regulation of growth rate and nucleic acid binding. The C26D10.5gene, targeted by miR-124, may have phospholipase A2 activity,which is involved in phosphorylation and post-embryonic bodymorphogenesis. These genes were only up-regulated under spaceradiation conditions, indicating that they might be affected byspace radiation with antagonistic effects of microgravity. Addition-ally, F18A12.8 and T01C4.2 are common target genes of miR-52,55 and 56, and these genes are related to neprilysin activity andbehaviour. These genes were down-regulated in the R+M and Renvironments, which indicates that they might affect changes inthe behaviour activities induced by space radiation.

These results suggest that space environmental stress can af-fect the expression of miRNA target genes. This occurrence mayregulate a variety of biological processes, such as embryonic devel-opment, reproduction, oviposition and positive growth regulationunder the space environment, including space radiation or micro-gravity.

3.3. Cel-miR-51 family miRNAs and target genes

It is known that the cel-miR-51 family of miRNAs (miR-51-56)functions in diverse regulatory pathways in C. elegans (Brenner etal., 2012). The miRNAs of the miR-51 family in C. elegans are char-acterised by a seed region (indicated by a black background inFig. 4). Compared with the sequence of miR-52, alignment analysisfor miR-55 and miR-56 showed 66.7% and 75% sequence similarity,respectively. This may explain why some genes can be commonlyregulated by the three members of the miR-51 family or are con-trolled by one or two members of the miR-51 family.

In the present study, we found that miR-52, 55 and 56 ofthe cel-miR-51 family were significantly up-regulated in the spaceenvironment. This indicates that their target genes were down-regulated to modulate biological processes. Based on different ex-pression patterns in the space synthetic and space radiation en-vironments, we analysed the putative target genes of each miRNA(Table 4). All of these predicted genes were shown to be expressedin the gene expression profile.

With regard to the cel-miR-52 targets, we found that theseputative genes might be involved in embryonic development, be-haviour, cell motion, oviposition and neprilysin activity. B0410.2and K11D9.1 were predicted to function in embryonic develop-ment, but these genes were not down-regulated in the spaceenvironment. To study the anti-correlated genes of miR-52, wesearched for corresponding targets that were down-regulated onlyin the R+M environment. R07E4.4 and C18A3.8, which function incell motion and behaviour, showed a slight down-regulation, whileT01C4.2b, C07H4.1 and F18A12.8b, which are possibly involved inbehaviour, membrane-related process and regulation of neprilysinactivity, were significantly down-regulated (> 2-fold changes) inthe R+M environment.

Furthermore, we analysed the putative targets of cel-miR-55and 56 and found that the biological categories of their targetgenes were similar to the miR-52 target genes. As for the alter-ation of the miR-56 target genes in both the R+M and R environ-ments, C54A12.1 (related to embryonic development) was slightly

down-regulated, while T01C4.1c and F18A12.8a, which are possi-bly involved in behaviour and the regulation of neprilysin activity,were significantly down-regulated (> 2-fold changes).

Notably, T01C4.2 and F18A12.8 genes are commonly targeted bymiR-52, 55 and 56. However, their isoforms could play a similarrole in different miRNA-regulated biological processes. T01C4.2band F18A12.8b contribute to miR-52 function, whereas T01C4.2cand F18A12.8a may contribute to miR-56 function.

In conclusion, we suppose that the three miRNAs of the miR-51family are sensitive to space environmental stress and could reg-ulate the biological responses of C. elegans through their potentialtarget genes.

4. Discussion

4.1. Altered miRNA expression in response to spaceflight

In the present study, we investigated the effect of spaceflight onthe miRNA expression profile of dauer C. elegans larvae during theSZ-8 mission. We analysed the changes in the miRNA expressionprofiles and predicted miRNA target genes, followed by miRNA-mRNA integration analysis. We reported that the space environ-ment could affect the miRNA expression profile of dauer larvae,and the altered miRNAs responded to spaceflight through the reg-ulation of multiple anti-corrected targets under the different spaceenvironments.

Considering the characteristics of C. elegans, we used the dauerlarvae as model organisms to study the effects of spaceflight onmiRNA expression profiles of space-flown C. elegans. It is knownthat the development of C. elegans consists of embryogenesis, fol-lowed by four larval stages (L1–L4) punctuated by moults. Dauerlarvae remain at a developmental stage equivalent to the L2-to-L3moult in response to environmental stimuli. It has been reportedthat the miRNA expression profile was dramatically changed at thedauer stage of C. elegans, which is involved in the regulation ofthe nematode’s developmental progress (Karp et al., 2011). There-fore, it is necessary to clarify whether the miRNAs altered in ourstudy are directly affected by the space environment or the dauerlife history. Comparative analysis of miRNA expression changesidentified 6 miRNAs that are affected by the dauer life history.Previous reports (Karp et al., 2011) identified a total of 14 miR-NAs. These miRNAs, including miR-84, 230, 237, 788, 799 and 795,were all down-regulated during the dauer stage. In contrast, ourresults showed that miR-237, 788, 799 and 795 were up-regulatedin either the space synthetic environment or the space radiationcondition, except for miR-84 and 230, which were down-regulated.Additionally, miR-52, 56 and 73 were unaffected by the dauer lifehistory and were up-regulated in the space environment in ourstudy (Table 2). These findings indicate that up-regulated miRNAsmay be affected by space synthetic factors, including space radia-tion and microgravity, in response to spaceflight.

4.2. Altered miRNA target genes in response to spaceflight

It is known that miRNAs act as critical regulators in gene ex-pression networks and play a key role in the cellular responseto environmental stresses. In our study, GO analysis was con-ducted on common putative target genes of miRNAs altered in thespace environment. The results showed that biological categoriescommon to the space synthetic and space radiation environmentswere those of “regulation of transcription”, “behaviour”, “oviposi-tion”, “positive regulation of locomotion”, “cell-cell signalling”, and“cell adhesion”. Most genes involved in embryonic development,post-embryonic development, nematode larval development andcellular protein localisation were enriched in the space syntheticenvironment. However, these categories were not enriched when

50 D. Xu et al. / Life Sciences in Space Research 1 (2014) 44–52

Table 4Analysis of putative target genes of altered miRNAs in cel-miR-51 family.

EnsemblGene ID

GeneSymbol

GO terms Space factorsa

R+M R

Cel-miR-52 1.79B0410.2 vang-1 embryonic development – –K11D9.1 klp-7 embryonic development, microtubule motor activity, – –C54D1.6 bar-1 behaviour, binding, oviposition – –C04D8.1 C04D8.1 Ras signalling −1.35 −1.50R07E4.4 R07E4.4 hydrolase activity, cell motion −1.34 −0.98C18A3.8 hlh-14 behaviour −1.33 −1.06T01C4.2b odr-2 behaviour −2.35 −1.62T01C4.2c odr-2 behaviour −2.64 −2.39C07H4.1 C07H4.1 membrane −2.53 −1.42F18A12.8a nep-11 neprilysin activity −3.15 −2.55F18A12.8b nep-11 neprilysin activity −2.08 −1.12

Cel-miR-55 1.91Y105E8A.16 rps-20 embryonic development, structural constituent of ribosome – –K11D9.1 klp-7 embryonic development, microtubule motor activity, – –C54D1.6 bar-1 behaviour, binding, oviposition – –C54A12.1 ptr-6 embryonic development, microtubule motor activity −1.32 −1.45R07E4.4 R07E4.4 hydrolase activity, cell motion −1.34 −0.98C18A3.8 hlh-14 behaviour −1.33 −1.06T01C4.2b odr-2 behaviour −2.35 −1.62T01C4.2c odr-2 behaviour −2.64 −2.39C07H4.1 C07H4.1 membrane −2.53 −1.42F18A12.8a nep-11 neprilysin activity −3.15 −2.55F18A12.8 nep-11 neprilysin activity −2.08 −1.12

Cel-miR-56 1.79 2.00K11D9.1 klp-7 embryonic development, microtubule motor activity, – –C54D1.6 bar-1 behaviour, binding, oviposition – –C54A12.1 ptr-6 embryonic development, microtubule motor activity, −1.32 −1.45R07E4.4 R07E4.4 hydrolase activity, cell motion −1.34 −0.98C18A3.8 hlh-14 behaviour −1.33 −1.06T01C4.2b odr-2 behaviour −2.35 −1.62T01C4.2c odr-2 behaviour −2.64 −2.39C07H4.1 C07H4.1 membrane −2.53 −1.42F18A12.8a nep-11 neprilysin activity −3.15 −2.55F18A12.8b nep-11 neprilysin activity −2.08 −1.12C08C3.1a egl-5 behaviour, DNA binding, oviposition −2.09 −0.99C08C3.1b egl-5 behaviour, DNA binding, oviposition −1.00 −0.46C08C3.1c egl-5 behaviour, DNA binding, oviposition −1.07 −0.77

Note: a All of listed genes are putative target genes of cel-miR-52, 55 and 56, which were also confirmed in analysis of gene expression profile. The expression of miRNAsand genes were presented by fold changes under space factors. “–” indicates that genes were not down-regulated. Those corresponding down-regulated mRNA target genesof miR-52, 55 and 56 were emphasised by bold words.

exposed to space radiation. In contrast, the biological categoriesin the space radiation environment were predicted to include theregulation of transcription, behaviours and oviposition. This is dueto the effect of microgravity on space synthetic factors becauseprevious work has reported that microgravity plays a role in mor-phology and the rate of development (Karp and Ambros, 2012;Karp et al., 2011). Additionally, we used dauer C. elegans larvae,which are at a different developmental stage, to identify a va-riety of space factors that could affect developmental processesafter the dauer stage, including embryonic development, post-embryonic development and nematode larval development, as wellas multicellular organism growth.

To improve the detection of functional miRNA-mRNA relation-ships, we analysed the C. elegans gene expression profiles, whichwere used to assess the miRNA expression levels. Prediction andintegration of miRNA target genes revealed that there were 12significant anti-correlated target genes targeted by 7 altered miR-NAs. These target genes are involved in a variety of biologicalprocesses, including embryonic development, locomotion, repro-duction, oviposition, DNA binding and the positive regulation ofgrowth rate in the space environment (Table 3). In particular, wefound that three members of the cel-miR-51 family and their tar-get genes changed in the space environment (Table 4). The miR-NAs of the miR-51 family in C. elegans are members of the old-est family of animal miRNAs described to date, with sequence

similarity among them. Previous work has shown that the cel-miR-51 family of miRNAs (miR-51-56), which functions in di-verse regulatory pathways in C. elegans, is required for embry-onic development and pharynx attachment (Brenner et al., 2012;Shaw et al., 2010). Here, we found that miR-52, 55 and 56 wereup-regulated in space conditions, resulting in the repression oftheir target mRNAs that might function in response to the spaceenvironment. The miR-52 target genes T01C4.2b, C07H4.1 andF18A12.8b, which function in behaviour, membrane, and neprilysinactivity, might play a role in the biological responses to micrograv-ity in the space synthetic environment. T01C4.2c and F18A12.8a,target genes of miR-56, were mainly affected by space radiation inthe space environment, indicating that different isoforms of geneswith identical functions could be targeted by different miRNAs inspecific environments. We suppose that the miRNA-mRNA changesmay translate into alterations of biological processes in the spaceradiation and/or microgravity environments.

It should be noted that the mRNA expression profile cannotreveal all of the miRNA target genes. It is known that miRNAsregulate gene expression by interfering with the protein transla-tional machinery and/or inducing the degradation of the targetmRNAs. In general, miRNAs repress protein translation at the post-transcriptional level by base pairing with the 3′-UTR, leading toreduced translation (Davis and Hata, 2009). Therefore, the mRNAlevel of miRNA target genes may not change in some cases. As

D. Xu et al. / Life Sciences in Space Research 1 (2014) 44–52 51

shown in Table 4, we found that some putative targets of miR-52,55 and 56 only exhibited a slight change (< 1.5-fold) or no changein mRNA expression, although these genes were shown to be ex-pressed in the mRNA expression profile analysis. Further studiesneed to be performed to investigate if the protein levels of themiRNA target genes change in the space environment.

4.3. The effects of microgravity and/or radiation on miRNA expression

Previous reports show that simulated microgravity and/or radi-ation could alter the expression of miRNAs in human lymphoblastcells (Mangala et al., 2011; Girardi et al., 2012). Of 352 miRNAs,7 miRNAs, including miR-22, 34a, 141, 150, 222, 618 and 423-5p(> 1.5-fold), were significantly up-regulated in simulated micro-gravity conditions. Analysis of the DNA microarray showed thatseveral transcription factors, such as EGR2, a putative target geneof miR-150, changed in simulated microgravity conditions, indi-cating a direct interaction with the altered miRNAs (Mangala etal., 2011). In the present study, we established two spaceflightgroups. This allowed us to compare between the two groups (SFvs. SC) to reflect the effects of microgravity on miRNA expres-sion in C. elegans. We found that the expression of several miR-NAs was significantly changed by microgravity, including miR-81,257, 795, 230, and 799 (data not shown). Of these, miR-257,miR-795, miR-230 showed increased expression, while miR-81 andmiR-799 showed decreased expression. Expression of miR-81 wasalso down-regulated in the space synthetic environment, whereasZK180.5, an anti-correlated gene of miR-81 with transcription fac-tor activity, was confirmed to be up-regulated in the space syn-thetic environment. These findings suggest that miRNAs can regu-late transcription factors in microgravity environments not only inhuman lymphoblast cells, but also in C. elegans.

Studies in different biological systems reported additive, syn-ergistic, antagonistic or independent interactions between radia-tion and microgravity (Canova et al., 2005; Girardi et al., 2012;Manti et al., 2005; Manti, 2006). It has been reported that miR-NAs act as post-transcriptional regulators of gene expression in theregulation of the DNA-damage response (DDR) to ionising radia-tion (IR) under modelled microgravity (MMG) in human peripheralblood lymphoblast cells (Girardi et al., 2012). MMG altered themiRNA expression profile of irradiated cells in a dose- and time-manner, with a decrease in the number of radio-responsive miR-NAs. Eight miRNAs (let-7i∗, miR-7, miR-7-1∗, miR-27a, miR-144,miR-200a, miR-598 and miR-650) were deregulated by the com-bined actions of IR and MMG. The ATM transcript was anti-correlated with miR-27a expression, which is associated with thep53 pathway of the DDR. Notably, pro-apoptotic BAX was anti-correlated with four miRNAs (miR-144, miR-200a, miR-598 andmiR-650) that can act together on an individual mRNA to produceadditive or synergistic effects in IR and MMG conditions. In ourstudy, space radiation and microgravity altered the miRNA expres-sion profile of C. elegans. Sixteen miRNAs were shown to be differ-entially expressed due to space radiation, among which miR-230and miR-799 were reversely affected by space radiation and micro-gravity, indicating the antagonistic interaction between radiationand microgravity. In contrast, both miR-52 and miR-56 were up-regulated and targeted C07H4.1, while both miR-81 and miR-82were down-regulated and acted on F55C5.4 in the space syn-thetic environment. This indicates that several miRNAs targetingone mRNA in C. elegans might also contribute to the production ofadditive or synergistic effects of radiation and microgravity. We didnot find any altered miRNA target genes that function in the DDRsignalling pathway of C. elegans in the R+M or R environment. Thismay be attributed to the different dose and energy features be-tween IR (high-dose, low LET) and space radiation (low-dose, highLET). The anti-correlated genes of the differentially expressed miR-

NAs were predicted to regulate reproduction, development, growth,behaviour and locomotion in the space environment, which mightbe related to the dauer larvae stage of C. elegans.

Dauer larvae are stress resistant and long-lived, which per-mits their survival in harsh conditions. Changes in physiologicaland biological activities at the dauer larvae stage are significantlydifferent from those in the normal developmental stages of ne-matodes. These changes involve energy metabolism, longevity, ge-nomic stability and behaviours (Burnell et al., 2005; Meyer etal., 2007; Wang and Kim, 2003). miRNAs affect gene expressionthrough a variety of mechanisms (Hammell et al., 2009; Holtz andPasquinelli, 2009), and a portion of the target genes can also havean impact on the expression of miRNAs. For example, the nuclearhormone receptor DAF-12, which is a target gene of let-7 andmiR-84, modulates the transcription of certain let-7-family miRNAsand miR-84 during developmental progression (Bethke et al., 2009;Hammell et al., 2009). It has been found that a feedback circuitinvolving let-7-family miRNAs and DAF-12 integrates environmen-tal signals and developmental timing in C. elegans (Hammell et al.,2009). We suppose that changes in the physiological and biologicalactivities of nematodes in a space environment can be controlledby miRNAs and their target genes, possibly forming a ”feedbackregulation loop” in the signalling pathway.

Acknowledgements

This work was supported by the National Natural Science Foun-dation of China (31270903) and the Fundamental Research Fundsfor the Central Universities (3132013327).

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