Effects of microgravity on DNA damage response in Caenorhabditis elegans during Shenzhou-8 spaceflight.

Abstract Purpose: Space radiations and microgravity both could cause DNA damage in cells, but the effects of microgravity on DNA damage response to space radiations are still controversial. Materials and methods: A mRNA microarray and microRNA micro- array in dauer larvae of Caenorhabditis elegans (C. elegans) that endured spaceflight environment and space radiations environment during 16.5-day Shenzhou-8 space mission was performed. Results: Twice as many transcripts significantly altered in the spaceflight environment than space radiations alone. The majority of alterations were related to protein amino acid dephosphorylation and histidine metabolic and catabolic processes. From about 900 genes related to DNA damage response, 38 differentially expressed genes were extracted; most of them differentially expressed under spaceflight environment but not space radiations, although the identical directions of alteration were observed in both cases. cel-miR-81, cel- miR-82, cel-miR-124 and cel-miR-795 were predicted to regulate DNA damage response through four different anti-correlated genes. Conclusions: Evidence was provided that, in the presence of space radiations, microgravity probably enhanced the DNA damage response in C. elegans by integrating the transcriptome and microRNome.


Introduction
During spacefl ight missions, crews endure both radiations and gravity regimens that profoundly diff er from those experienced on Earth (Horneck 1999, Manti 2006. Space radiations have been characterized as high linear energy transfer (LET) and low dose rate, which may induce serious DNA damage over a wide spectrum when compared with radiation exposure on the ground (Ohnishi et al. 2002, Cucinotta and Durante 2006, Dziegielewski et al. 2010. Cells have evolved highly conserved DNA repair machinery to process the damage by an effi cient DNA damage response (DDR), involving the activation of cell cycle checkpoints to induce a cell cycle arrest, DNA repair, apoptosis, or a combination thereof (Harper and Elledge 2007). Th e transcriptional profi le also changes in response to DNA damage, probably in order to promote cell survival (Abbotts et al. 2014). Disrupting DDR may elicit genome instability and increasing risks of carcinogenesis (Cucinotta et al. 2001, Cucinotta and Durante 2006, Hei et al. 2011. Microgravity is another inevitably risk factor to health during spacefl ight, which could impair signal transduction and immune response (Morrison 1994, Aubert et al. 2005. Simulated microgravity is reported to aff ect the expression of genes involved in DNA repair, resulting in accumulation of DNA damage (Takahashi et al. 2012). Studies on the combined eff ects of microgravity and radiation have had confl icting results, with some showing that microgravity or simulated microgravity increased the DNA damage induced by ionizing radiation (IR) (Degan et al. 2005, Kumari et al. 2009, Wang et al. 2011, Yatagai et al. 2011, others showing they decreased the damage (Kobayashi et al. 2000(Kobayashi et al. , 2004, and some showing they had no eff ects (Pross et al. 2000, Mognato andCelotti 2005). Th erefore, whether real microgravity can aff ect the DDR to space radiations is still unclear.
MicroRNA (miRNA), a class of small non-coding RNA, mediates post-transcriptional regulation of specifi c target mRNA in various cellular processes (Bartel 2009). Recently, miRNA has been shown in regulating a variety of physiological activities such as DDR process under microgravity and/or radiation environments (Kato et al. 2009, Mangala et al. 2011, Wouters et al. 2011). Girardi reported that simulated microgravity altered the miRNA expression signature of irradiated cells by decreasing the quantity of radio-responsive miRNA. Genes in the Gene Ontology (GO) category ' Response to DNA damage stimulus ' were enriched under 1 g but not microgravity conditions, indicating that simulated microgravity could aff ect the DDR process to IR in human lymphocytes (Girardi et al. 2012). In our previous study, 23 altered miRNA of Caenorhabditis elegans showed diff erent expression patterns under spacefl ight environment and space radiations environment, and seven miRNA were predicted to regulate 12 genes by integration analysis of the miRNA and mRNA expression profi les (Xu et al. 2014). Th erefore, miRNA and their target genes could be involved in the DDR process of living organisms when exposed to space environment.
C. elegans is simpler than the mammalian system while still sharing high genomic homology and thus employed for space biological studies as an excellent model . Under spacefl ight environment, the sensitivity of physiological processes in C. elegans varied from endpoints to endpoints, such as developmental timing (Zhao et al. 2005, locomotion (Oczypok et al. 2012), apoptosis (Higashitani et al. 2005), mutant rate (Zhao et al. 2006), muscle development (Higashibata et al. 2006, and aging (Honda et al. 2012). However, it was not clear whether these changes were generated by space environment or by metagenesis during the space missions, because larvae used in most studies could keep breeding during spacefl ight. To avoid the infl uence from metagenesis (Morey-Holton et al. 2007), an eff ective solution would be chosen a diapause stage to maintain C. elegans worms in a synchronized stage. Th e dauer diapause stage of C. elegans is a stress-resistant stage characterized by no feeding, lifespan extending and developmental arrest (Wang and Kim 2003, Hu 2007, Jeong et al. 2009). Hence, dauer larvae were employed in our study.
To elucidate the eff ects of microgravity on the DDR process in the presence of space radiations, transcriptome and microR-Nome of space-fl own C. elegans were integrated to analyze gene expression and the corresponding miRNA expression. Th e genes involved in the processes of DNA repair, apoptosis, cell cycle arrest, and others related to DDR process, were focused on to search for a possible miRNA regulating gene expression in the dauer larvae of C. elegans fl own on Shenzhou-8 spacefl ight.

Sample preparation of C. elegans
Th e wild-type strain ( Bristol N2 ) of C. elegans was acquired from the Caenorhabditis Genetics Center (Minneapolis, MN, USA). Worms were cultured on Nematode Growth Medium (NGM) at 23 ° C and synchronized to dauer larvae by 1% sodium dodecyl sulfonate (SDS) (Sangon Biotec, Shanghai, China) according to the protocols in Wormbook (Stiernagle 2006). C. elegans were divided into three groups as shown in Table I: spacefl ight group, spacefl ight control group, and ground control group. As described in previous studies (Horneck 1999, Takahashi et al. 2012, experiments were designed to obtain biological eff ects concerning space radiations (SR) by comparing results between spacefl ight control group and ground control group (group 2 vs. 3), and to obtain biological eff ects concerning spacefl ight environment (SF) by comparing results between spacefl ight group and ground control group (group 1 vs. 3).

Spacefl ight experiments
Shenzhou-8 was launched in the Jiuquan Satellite Launch Center on 1 November 2011. About 10 h before the launch, fresh solid NGM and approximately 10 5 dauer worms were successively loaded in the Experiment Unique Equipment (Supplementary Figure 1A, available online at http://informahealthcare.com/ abs/doi/10.3109/09553002.2015.1043754 available online), which was housed in the Experiment Containers (Supplementary Figure 1B available online at http://informahealthcare.com/ abs/doi/10.3109/09553002.2015.1043754). Assembled containers were settled into the centrifugal and static slots of BIOBOX (Supplementary Figure 1C and D available online at http://informahealthcare.com/abs/doi/10.3109/09553002. 2015.1043754). Th e Shenzhou-8 mission was fl own from 1 -17 November 2011, with a total mission time of 16.5 days. Th roughout the duration of the experiment, the Experiment Containers maintained a temperature of 23 Ϯ 0.5 ° C. G-force peaks did not exceed levels of Ϯ 0.01 g , while most of the time values oscillated between Ϯ 0.005 g . Space radiations dose was measured by the thermoluminescent detector (TLD) in the static slot (1.92 mGy) and centrifuge slot (2.27 mGy), respectively. Seven hours after landing, worms were collected and kept in liquid nitrogen. Th e corresponding ground controls of the experiment were performed in parallel at the Payload Integration Test Center in Beijing 2 days later. Here, the samples were kept in static slot in BIOBOX and temperature was maintained at 23 Ϯ 0.5 ° C. Th e ground group was stopped after 16.5 days and the samples were collected after 7 h later. Finally, worms were sent back to Dalian Maritime University for further analysis.

Total RNA isolation
About 2000 worms from each group were collected and total RNA was isolated using Invitrogen ™ TRIzol (Invitrogen, Carlsbad, CA, USA) according to manufacturers ' instructions. Quality and purity of the RNA preparations were assessed by spectrophotometric determination (NanoDrop ® 2000c UV-Vis Spectrophotometer) (Th ermo Fisher Scientifi c Inc., Wilmington, DE, USA) of the ratio of absorbance at 260/280 nm (OD 260 / OD 280 Ͼ 1.9) and by quantifi cation of the ratios of 28S:18S ribosomal RNA (GelDoc-ItTM 310 Imaging System) (UVP, Cambridge, CA, USA).

mRNA microarray and miRNA microarray analysis
Th e NimbleGen Gene Expression Profi ling service and miRCURY ™ LNA Array microRNA Expression Profi ling service were performed by KangChen Bio-tech Inc. (Shanghai, China) as previously described (Xu et al. 2014). GO analysis was applied to determine the biological process of genes. Genes involving in DDR process belong to categories of DNA repair (GO: 0006281), apoptotic process (GO: 0006915), cell cycle arrest (GO: 0007050), response to DNA damage stimulus (GO: 0006974), telomere maintenance (GO:0000723) and other processes involved in DDR according to the database from AmiGO (version 1.8) (Ashburner et al. 2000), KEGG pathway, and Wormbook (O ' Neil and Rose 2006). To predict the target miRNA of differentially expressed genes, computational analysis was performed with combination of miRanda (Betel et al. 2008), MicroCosm Targets (Version 5), and TargetScanWorm 6.2.

Quantitative real-time polymerase chain reaction analysis
Th e data of mRNA microarray were validated by quantitative real-time polymerase chain reaction (qRT-PCR) using Super-Script ® III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and SYBR ® Green Master Mix (Applied Biosystems, Foster City, CA, USA). Total RNA was extracted from independent worm samples for qRT-PCR. Th e reactions were incubated in an ABI PRISM7900 HT system (Applied Biosystems, Foster City, Eff ects of microgravity on DNA damage response 533 CA, USA) in PCR plates (Applied Biosystems, Foster City, CA, USA) for 10 min at 95 ° C, followed by 40 cycles of 10 s at 95 ° C and 60 s at 60 ° C. Results were normalized to the threshold cycle (Ct) value of house-keeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ( gpd-2 ), and the relative expression levels of genes were calculated using the 2 -Δ Δ Ct method. Primers were shown in Supplementary Table I

Statistical analysis
Raw data were log2 -transformed to achieve the statistical analysis under MS Excel 2010 software. R version 2.15.2 software was used to acquire the features of the datasets. Diff erentially expressed mRNAs (log2-fold changes Յ Ϫ 1 or Ն 1) and miRNA (log 2-fold change Յ Ϫ 1 or Ն 1) were identifi ed through fold change fi ltering. Cluster 3.0 version 1.24 and Java TreeView version 1.0.4 were used to achieve hierarchical clusters of diff erentially expressed miRNA.

Global gene expression profi le under diff erent space environments
mRNA microarray analysis showed that 2262 transcripts changed among the 18,186 detected transcripts under SF environment. By contrast, only half the number of altered transcripts (1078) were observed under SR environment ( Figure 1A). Th e percentage of up-regulated transcripts and down-regulated transcripts appeared nearly to be equal in both the SF and SR environments ( Figure 1B). Th e distribution characteristics of global gene expression profi le were evaluated by the interquartile range (IQR). Th e value of IQR under SF environment (0.827) was higher than that under SR environment (0.621), indicating that more genes were signifi cantly altered in response to SF environment ( Figure 1C and 1D). Diff erentially expressed transcripts were subjected to GO analysis as shown in Table II. In the all of biological processes, ' protein amino acid dephosphorylation ' represented the largest group of up-regulated genes, followed by ' dephosphorylation ' , ' post-translational protein modifi cation ' , and so on under SF environment. ' Histidine metabolic process ' , ' histidine catabolic process ' , ' histidine family amino acid metabolic process ' , and ' histidine family amino acid catabolic process ' represent important groups of down-regulated genes under SF environment. Under SR environment, diff erentially expressed genes were the most enriched in ' polyol metabolic process ' , ' acyl-CoA metabolic process ' and ' thioester metabolic process ' .

Expression profi le of genes involving in DDR process
About 900 genes involved in DDR process were screened out (Figure 2), and of those, 38 diff erentially expressed genes 1.5   (Table IV).

Microarray data validation by qRT-PCR
Microarray data from mRNA expression profi ling were validated by qRT-PCR from independently-isolated RNA samples ( Figure 3). Ten genes were selected from diff erentially expressed genes in microarray, including H21P03.2, exo-3 , him-6 , air-2 , cdh-3 , dyf-2 , sir-2.2 , hda-4 and Y56A3A.33. Th e direction and amplitude of the fold changes determined by qRT-PCR closely matched microarray data for genes tested were extracted under SF and SR environments (Table III). Under SF environment, 31 genes were altered, including seven in DNA repair, 17 in the apoptosis process, and the rest were altered in various other processes involved in DDR process. In contrast, four diff erentially expressed genes were observed under SR environment, including two in ' DNA repair ' and two in ' apoptosis process ' . Besides, agt-1 , him-6 in ' DNA repair ' and T05C3.6 in ' apoptosis ' were altered in both cases. Genes in cell cycle arrest (GO: 0007050) were found to have no change in both cases (data not shown).  Figure 3. Microarray data validation by quantitative real-time PCR (qRT-PCR). qRT-PCR was used to confi rm the diff erential expression of 10 genes identifi ed by microarray: him-6 and air-2 are up-regulated, dyf-2 and sir-2.2 are down-regulated, H21P03.2, exo-3 , hda-4 and Y56A3A.33 are no change under SF environment. him-6 and cdh-3 is up-regulated under SR environment. Fold changes is determined using the 2 -Δ Δ Ct relative quantitation method with GAPDH ( gpd-2 ) as the endogenous control gene.

Discussion
Space radiations and microgravity could induce DDR in living organisms, while the interaction between them is not determined. Th e combined infl uence of space radiations and microgravity with which humans in space have to cope must be taken into consideration. In this study, an analysis of mRNA microarray and miRNA microarray was performed to elucidate the eff ects of microgravity on the DDR process of space-fl own C. elegans in the presence of space radiations. Th e cellular responses invoked by DNA damage consist of a broad network of transcriptionally regulated pathways covering most aspects of cellular physiology, such as cellular signaling, metabolic pathways and protein modifi cation (Jen andCheung 2003, Forrester et al. 2012). In this study, the global gene expression profi les under diff erent experimental conditions were analyzed in order to study the eff ect of microgravity on DDR process in the presence of space radiations. Results showed the number of diff erentially expressed genes under SF environment was about double that under SR environment, and altered genes appeared to have much more obvious changes under SF environment than SR environment (Figure 1). GO analysis showed that altered genes under SR environment were enriched in six categories (Table II). In animals, acetyl-CoA is mainly produced from fatty acid metabolism and is essential for lipid metabolism. Polyol metabolic process is probably related to worm energy supply (Blaise et al. 2007). Hence, space radiations may lead to a shift in energy metabolic homeostasis during spacefl ight (Mao et al. 2014), which probably is a relevant adjustment to space environmental stress in C. elegans at dauer or developmental stage (Selch et al. 2008). In contrast, a large number of altered genes function in protein metabolic processes and modifi cation under SF environment. Genes in metabolic and catabolic processes of histidine and related amino acid may be involved in antioxidant eff ect (Farshid et al. 2013) and resistance to metal toxicology (Murphy et al. 2011). Protein amino acid dephosphorylation and phosphorylation changes have functional pluripotency in almost all physiological activities induced by radiation or reduced gravity, such as signal transduction (Tauber et al. 2013), transcription (Trivigno et al. 2013), endothelial dysfunction (Versari et al. 2013), muscle damage/recovery (Shtifman et al. 2013). Higashibata also found that expression of phosphoprotein changed signifi cantly in space-fl own C. elegans (Higashibata et al. 2007). Th erefore, the fi ndings indicated that microgravity could aff ect transcriptional modulation involved in DDR under spacefl ight environment.
DDR is a complex pathway addressed to maintain genome integrity through the activation eff ector proteins of DNA repair, apoptosis and cell cycle arrest (Girardi et al. 2012). DDR to IR depends on the LET and the dose of radiation,  Under SF environment, ung-1 , exo-3 and pme-1 possessing nuclease activity in Base excision repair (BER) or Nucleotide excision repair (NER) pathways were altered, and polk-1 acting as DNA polymerase in Fanconi anemia (FA) process was altered as well. Under SR environment, the expressions of lig-4 encoding DNA ligase in NHEJ and F35H10.5 in FA pathway were changed. Th e expression pattern of him-6 and agt-1 indicated the enzymes being increased under SR environment, whether combination with microgravity or not. Th e results indicated that cells probably activated more thus space radiations consisting of complex particles may induce a variety of DNA damage, such as single strand DNA lesions (SSL) or double-strands DNA breaks (DSB) (Hada andGeorgakilas 2008, Lemmens andTijsterman 2011  expressions of miRNA have been reported in response to radiation or microgravity. For example, the miR-34 is required for the DDR in vivo in C. elegans and in vitro in human breast cancer cells (Kato et al. 2009). In human lymphocytes, eight miRNA were dysregulated by the combined action of IR and simulated microgravity in another study (Girardi et al. 2012). We found that miRNA expression showed diff erent patterns under SF environment and SR environment. Furthermore, miR-81/82, 124 and 795 were likely to regulate DDR process by targeting air-2 , bath-41 , pme-1 and daf-16, respectively (Table IV). Th e cel-miR-81/82 family is partial homology to hsa-miR-143 (Lim et al. 2003). Hsa-miR-143, widely down regulating in many cancer cells, has been reported to protect cells from DNA damage-induced killing (Lin et al. 2011), and inhibit tumor cell growth of gastric cancer , breast carcinoma (Ng et al. 2014) and colorectal cancers (Pagliuca et al. 2013). Hence, down-regulated miR-81/82 in space-fl own C. elegans implied the possible risk of tumors under space environment. miR-124 is a highly conserved miRNA proven to regulate DNA repair protein Ku70 in rats brain (Zhu et al. 2013), radiosensitize human glioma cells by targeting CDK4 (Deng et al. 2013), and inhibit Reactive oxygen species (ROS) formation and aging in C. elegans (Dallaire et al. 2012). Given that potential DDR from space radiations exposure results from direct DNA break or indirectly from the production of ROS (Cucinotta and Durante 2006), miR-124 was possible to regulate DDR by multiple pathways in response to spacefl ight. miR-795 is speculated to promote apoptosis by decreasing daf-16 (Perrin et al. 2013), while little is known about miR-795. Th ese results indicated that miRNA might be involved in the DDR process in space-fl own C. elegans .
To disentangle the complex interplay of the parameters of spacefl ight environment, a set of appropriate control experiments in space and on the ground are required. As reported widely in previous studies (Horneck et al. 2010, Takahashi et al. 2012, the use of an on-orbit 1 g centrifuge can provide an ideal method for ensuring that the experimental groups are exposed to the same overall space environmental factors with the exception of g -level. Th e approach permits a separate determination of the eff ects of radiation or microgravity, as well as interactions of both parameters of space (Horneck 1999). Given that the space radiations were always present during our practical spacefl ight, we discussed the synergistic eff ects of microgravity by comparing the pathways to repair DNA lesions under SF environment compared with SR environment. Microgravity also probably infl uenced the genomic stability by telomere maintenance because three genes were altered under SF environment. gei-17 , encoding the small ubiquitin-like modifi er (SUMO) E3 ligase, is required for telomere anchoring in C. elegans (Ferreira et al. 2013). pif-1 encodes a conserved DNA helicase of the PIF1 subfamily, which negatively regulates telomerase activity that acts by displacing telomerase from the ends of DNA (Boule et al. 2005, Eki et al. 2007). C11G6.2 is involved in telomere maintenance by GO identifi cation.
Apoptosis is essential for the development and survival of most multicellular animals by preventing growth of cells mutation due to DNA damage (Lettre and Hengartner 2006). Th e pathways leading to DNA damage-induced apoptosis are surprisingly complex, although several members in core pathway have been identifi ed in C. elegans , including cep-1 , ced-9 , ced-4 and ced-3 (Lord and Gunawardena 2012). In our results, p53 like gene cep-1 in known core apoptosis pathway (Salinas et al. 2006, Greiss et al. 2008 was not observed to change. None of genes in CED gene family changed under space environments (data not shown), which may attribute to the occurrence of normal apoptosis in space-fl own C. elegans as reported in previous study (Higashitani et al. 2005). However, 20 altered genes associated with apoptosis in response to spacefl ight, such as positive ( dct-17 , pal-1 , dct-1 ), negative ( eya-1 ) or other ( cccp-1 ) regulators, receptor-mediated endocytosis ( pdcd-2 , F43D9.3, hsp-1 ) were found. Most of apoptotic genes were regulated under SF environment (17/20), while their expression profi le did not show any obviously tendency to function in apoptosis. Alteration of these genes may be an accommodation in response to space radiations and microgravity (Kumari et al. 2009), and further study is needed.
Moving on from the biological processes of DNA repair, apoptosis, and cell cycle arrest, other changes related to DDR were detected as well, such as response to DNA damage stimulus (Y56A3A.33, daf-16 sir-2.2 ), chromosome structure ( hda-4 ), cytokinesis checkpoint ( air-1 , sep-1 ), and telomere maintenance ( trt-1 ). Th ese genes were altered under the SF environment only, also indicating that microgravity probably enhanced DDR in the presence of space radiations.
miRNA plays an important role in a variety of biological processes by negatively regulating gene expression. Aberrant diff erence between the responses to spacefl ight with and without microgravity, and found that microgravity involved in several biological processes on gene expression and miRNA expression. Interestingly, most of genes involved in DDR processes signifi cantly altered under SF environment but not SR environment, although the identical directions of alteration of genes were observed in both cases. Th ese might result from a better resistance to ROS and genomic instability in dauer larvae (Burnell et al. 2005, Ruzanov et al. 2007). More possibly, the results indicated the limited impact of space radiations encountered during a 16.5-day spacefl ight mission.
Although several studies showed the eff ects of low-dose radiation (Hartman et al. 2001, Maalouf et al. 2011, Takahashi et al. 2012, Schenten et al. 2013, proper long duration space missions are still needed for studying the interaction of space radiation and microgravity. In this study, gene expression profi le and miRNA expression profi le in dauer larvae of C. elegans during Shenzhou-8 spacefl ight mission were analyzed. Results indicate that during the short-duration spacefl ight, microgravity probably enhanced the biological response on transcription and posttranscriptional regulation, in particular on DDR process, and these fi ndings suggest how safe or hazardous the radiation combined with microgravity exposure is for the astronauts, which may be helpful for space risk assessment.