Effects of simulated microgravity on microRNA and mRNA expression profile of rat soleus

Acta Astronautica 107 (2015) 40–49

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Effects of simulated microgravity on microRNA and mRNA expression profile of rat soleus Hongjie Xu a,b, Feng Wu b, Hongqing Cao b, Guanghan Kan b, Hongyu Zhang b, Ella W. Yeung c, Peng Shang a, Zhongquan Dai b,n, Yinghui Li a,b,n a

School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, China State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, Beijing 100094, China c Muscle Physiology Laboratory, Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong b

a r t i c l e in f o

abstract

Article history: Received 3 August 2014 Received in revised form 16 October 2014 Accepted 16 November 2014 Available online 21 November 2014

Spaceflight induces muscle atrophy but mechanism is not well understood. Here, we quantified microRNAs (miRNAs) and mRNA shifts of rat soleus in response to microgravity. MiRNAs and mRNA microarray of soleus after tail suspension (TS) for 7 and 14 days were performed followed by target gene and function annotation analysis and qRT-PCR. Relative muscle mass lost by 37.0% in TS-7 but less than 10% in the following three weeks. TS altered 23 miRNAs and 1313 mRNAs with at least 2-fold. QRT-PCR confirmed some of these changes. MiR-214, miR-486-5p and miR-221 continuously decreased. MiR674 and Let-7e decreased only in TS-7, while miR-320b and miR-187 decreased only in TS14. But there was no alteration of miR-320 and miR-206 in both time point. For mRNA detection, actn3 (5.1-fold and 13.8-fold) and myh4 (38-fold and 51.6-fold) increased abundantly and a3galt2 decreased. Predicted targeted genes (whyz, ywhaz and SFRP2) of altered miRNAs decreased. GO terms and cellular pathway of these alteration showed enrichment in regulation of muscle metabolism. Integration analysis of the miRNA and mRNA expression profiles confirmed that eleven genes were differently regulated by four miRNAs. This is the first study that showed expression pattern and synergistical regulation of miRNA and mRNA in rat soleus of TS for up to 14 days. & 2014 IAA. Published by Elsevier Ltd. All rights reserved.

Keywords: Tail suspension MicroRNAs Skeletal muscle atrophy Simulated microgravity Microarray

1. Introduction Spaceflight causes several adaptations of skeletal muscle, including fiber atrophy/wasting and a slow to fast shift,

especially in weight-bearing soleus [1,2]. Although diverse exercise countermeasures have been explored for space muscle atrophy [3–7], an efficient approach remains a longstanding challenge in space medicine. The exact mechanism

Abbreviations: miRNAs, microRNAs; TS, tail suspension; SMG, simulated microgravity; IGF-1, insulin-like growth factor 1; PI3K/Akt, phosphatidylinositol 3-kinases/protein kinase B; PTEN, phosphatase and tensin homology deleted on chromosome 10; SFRP2, Secreted frizzled-related protein 2; ywhaz, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta; whyz (zfhx), zinc finger homeobox gene x; MAPK, Mitogen-activated protein kinases; DMD, Duchenne muscular dystrophy; IRS, insulin receptor substrate; MHC, myosin heavy chain; Myh1, Myosin1 also known as myosin, heavy chain 4 n Corresponding author at: China Astronaut Research and Training Center, State Key Laboratory of Space Medicine Fundamentals and Application, P.O.Box. 5132-23, No. 26 Beiqing Road, Haidian District, 100094, Beijing, China. Tel.: þ 8613683338750; fax: þ 861068117399. E-mail addresses: [email protected] (Z. Dai), [email protected] (Y. Li). http://dx.doi.org/10.1016/j.actaastro.2014.11.021 0094-5765/& 2014 IAA. Published by Elsevier Ltd. All rights reserved.

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of muscle atrophy induced by microgravity is not well understood till now. Skeletal muscle atrophy was defined as a decrease of muscle mass and occurs when protein degradation exceeds protein synthesis [8]. Major advances have been witnessed in the understanding of muscle diseases and significant inroads are being made to muscle atrophy treatment in the last 20 years. Excellent reviews have been published on cellular and molecular mechanism of muscle atrophy [8,9]. Notably, IGF-1 is central due to its unrivaled pleiotropic ability [8] and its downstream PI3K/Akt that controls both protein synthesis and degradation. Despite immense progresses made using several approaches, no curative therapy is currently available for any of muscle atrophy including space muscle atrophy and only palliative and symptomatic treatment is available for patients [9]. Recent advancements in the field of molecular biology have revealed that non-coding small RNAs ( 21 nt) have broad effects on regulatory gene expression networks through targeting complementary mRNAs which leads to mRNA degradation [10,11]. Increased data demonstrated that miRNAs play a pivotal role for muscle development and regeneration. MiRNAs expression was detected during the 18 developmental stages of porcine longissimus muscle by Solexa sequencing technology and revealed that some miRNAs, such as miR-378, miR-1 and miR-206, abundantly expressed in skeletal muscles [12]. Recent reports showed that miR-206 and miR-486-5p play essential roles in muscle fiber development, plasticity, metabolism and myogenesis [13,14]. Some miRNAs were able to directly and/or indirectly modulate cytokines, hormones and signaling molecules which were related to muscle atrophy [11]. MiR-320 interacted with Wnt/ beta-catenin and insulin-PI3K signaling pathway [15,16]. Skeletal muscle enriched miR-486-5p has been demonstrated to target PI3K inhibitor PTEN, thus activating Akt pathway [17]. On the other side, regulation of these miRNAs was controlled by key myogenic regulatory factors (MRFs) and influenced by microenvironments such as mechanical stress [18]. These insights raised a compelling rationale to target miRNAs for mechanism investigation or countermeasure against muscle atrophy induced by microgravity. Limited data showed that miRNAs could response to simulated microgravity (SMG) in plant solanum [19], human lymphoblastoid cell [20] and rat gastrocnemius muscle [21]. Tail suspension/hind-limb suspension is the primary animal model to simulate microgravity. In present study, we aimed to explore the synchronous changes of mRNA and miRNAs expression profile in soleus response to tail suspension (TS). The results confirmed several miRNAs alteration accompanied muscle atrophy induced by TS, which would enhance the understanding of space muscle atrophy and the function of miRNAs during their responsiveness to mechanical stress. 2. Materials and methods 2.1. Rat tail suspension Animal work was performed with the approval from the ethics committee of China Astronaut Research and Training Center. Adult male Sprague-Dawley (SD) rats (200–250 g and eight weeks old; Vital River, Beijing, China) were housed in

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cages on a routine 12 h light/dark cycle with ad libitum access to food and water under controlled (2272 1C) conditions, and acclimatized for one week before the experiment. A total of 82 rats were studied. Animals were randomly divided into six groups: control for seven days (CN-7), control for 14 days (CN-14), control for 28 days (CN28); tail suspension for seven days (TS-7), tail suspension for 14 days (TS-14) and tail suspension for 28 days (TS-28) (n¼16 for 7- and 14-day group, n¼9 for 28-day group). Rats were suspended at an approximately 301 angle by taping its tail to obtain hindlimb unloading condition other than CN group as previously described methods [22]. All surgery was performed under sodium pentobarbital (1%, 1 mL/100 g weight, i.p.) anesthesia and then sacrificed, and all efforts were made to minimize suffering. At the experiment time point, soleus muscle was dissected out, weighed and snap frozen in liquid nitrogen till RNA extraction. 2.2. Total RNA and miRNAs isolation Total RNA and miRNAs were isolated from rats soleus muscle by RNAiso plus and RNAiso plus for miRNAs (Takara, Dalian, China) respectively followed manufacturer’s protocol. Quality and purity of RNA were assessed by the ratio of OD260 and OD280, which of the value of all samples ranged from 1.8 to 2.2. RNA integrity assess used the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). The RIN (RNA integrity number) value of all samples ranged from 8.1–8.9 (scale 1–10), indicating high-quality RNA. 2.3. Affymetrix GeneChips assay and dada analysis MiRNAs and mRNA expression profiles were determined in three groups: control (CN-7), TS-7 and TS-14. Affymetrix GeneChips Rat Genome 230 2.0 Array and Affymetrix GeneChip miRNA Array according to Sanger miRBase V11.0 were respectively used for mRNA and miRNA expression profile analysis. Mixture of four samples from each group was detected by gene chip carried out by oebiotech inc. (Shanghai, China) following manufacturer’s standard instruction. RNA sample was purified further using the RNeasy mini Kit (Qiagen, Germany). For miRNA array, PAP Enzyme was used for poly(A) tailing followed by label and hybridization using Flash Tag Biotin for Affymetrix miRNA arrays (Genisphere, USA) and GeneChips Expression 30 Amplification ReagentsHybridization Controls (Affymetrix, USA). For mRNA array, in vitro transcription was done to synthesize biotin-labeled cRNA using 3' IVT Express Kit (Affymetrix, USA) followed by hybridization using GeneChips Hybridization. All samples were washed and stained using GeneChips Wash and Stain Kit, then scanned, transferred into original data, and analyzed for fold change (fc) by GeneSpring 10 software. The probesets in miRNA array sharing the same sequence of different species were considered as one and average value of these probe-sets were analyzed. That fc Z2 was taken as requirement of significant change. Microarray data (CEL files) were also deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus repository under the accession number GSE52057. Targeted genes of changed miRNAs were predicted using TargetScan (http:// www.targetscan.org/) database. 1614 and 1991 probe-sets ID

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with at least 2-fold difference in mRNA chip of TS-7 and TS14 respectively were integrated into DAVID v6.7 (http://david. abcc.ncifcrf.gov/home.jsp) to analyze the gene ontology and function annotation [23]. 2.4. Validation of selected genes using quantitative PCR Quantitative realtime RT-PCR (qRT-PCR) was used to validate changes of mRNA and miRNAs expression observed in the microarray, as well as predicted targeted genes of altered miRNAs. First-strand cDNA was prepared from total RNA (1 μg) by PrimeScriptTM RT reagent Kit With gDNA Eraser for mRNA and One Step PrimerScriptTM miRNA cDNA Synthesis Kit (Perfect Real Time) (Takara, Dalian China) for miRNAs according to the manufacturer’s specifications. 1 μL of 5-fold dilution of cDNA and 0.4 μM of primer pair (Supplemental Table S1) were used in 20 μL reaction volume with SYBRs Premix Ex Taq II (Perfect Real Time) (Takara, Dalian China) in mastercycler realplex (Eppendorf, Germany). These qRT-PCR procedures were run in duplicate to correct for variances in loading. All PCR results were determined using the relative ΔΔ quantification method (2- Ct) [24] with GADPH or small nuclear U6 RNA (snRNA U6) as the normalization control. 2.5. Statistical analysis Data were presented by the mean 7S.D. with significant difference between two groups used by independent Student’s t test. Muscle mass results were analyzed by One-way ANOVA to identify significant differences using SPSS 19.0 software, with Po0.05 taken as statistical difference, and Po0.01 as significant difference. 3. Results

Furthermore, there was a slight trend of decrease among three TS groups without statistical difference, implying that the speed of mass loss stepped down. The same pattern was observed in both soleus and medial gastrocnemius atrophy in all three models to simulate microgravity namely spaceflight, TS and spinal cord isolation [26]. Additionally, muscle relative mass of CN-28 decreased slightly but no statistical difference when compared with CN-7 or CN-14. 3.2. Altered miRNAs expression response to tail suspension To identify microgravity-responsive miRNAs, miRNA expression profile of rat soleus after TS for 7 and 14 days was measured using Affymetrix GeneChip miRNA Array. 1801 probe-sets of 7626 (174 of 351 rat miRNAs) were detected in the normal rat soleus muscle. As expected, the muscle-specific miRNAs (miR-206, miR-1 and miR-133) were some of the most abundant miRNAs, as well as Let-7, miR-23 and miR-24 family. Compared with CN group, the expression of 23 rat miRNAs changed at least 2-fold in TS-7 and/or TS-14 (Table 1). These miRNAs exhibited three expression patterns that were changed respectively in TS-7, TS-14 and both. QRT-PCR was performed to validate the effect of TS, including six miRNAs related to muscle in Table 1 and the other three with either abundantly expressing (miR-206) or specific function in skeletal muscle response to gravity (miR214 and miR-221) [13,27,28]. The result showed that miR214 ( 1.97), miR-486-5p ( 1.92), miR-221 ( 1.60), miR-674 ( 1.41) and Let-7e (1.35) changed in TS-7, and that miR214 ( 2.05), miR-486-5p (1.92), miR-221 ( 2.67), miR320b ( 2.58) and miR-187 ( 1.69) changed in TS-14. Of these showed three expression patterns. Almost all results were corresponding to that of the microarray (Fig. 2).

3.1. Muscle atrophy induced by tail suspension

3.3. Effect of tail suspension on mRNA expression profile

Analysis of soleus muscle mass normalized to body weight revealed that muscle relative mass lost 37.0% in TS-7, 46.7% in TS-14 and 52.6% in TS-28 (Fig. 1). This was consistent with previous study that reported a decrease in TS-7, however without change of two days TS [25].

Because miRNAs influence gene expression, we further checked whether alteration of miRNAs was correlated with mRNA expression by performing mRNA microarray, of which 51.09%, 53.80% and 56.06% of 31000 probe-sets were detected in CN, TS-7 and TS-14 respectively. The result Table 1 List of miRNAs that changed at least 2-fold in microarray in TS-7 and/or TS-14a. Pattern 1

Fig. 1. Effect of tail suspension (TS) on rat soleus muscle relative mass. Soleus muscles of all animals were dissected, weighed immediately and normalized to body weight. (n¼ 9 of TS-28 and n ¼16 of others, nnPo 0.01).

Pattern 2

Pattern 3

miRNA

TS-7

TS-14

miRNA

TS-7

miRNA

TS-14

miR-320a miR-320b miR-320c miR-1826 miR-674 miR-1308 miR-709 miR-923

2.31 2.03 2.33 2.01 2.37 2.40 2.67 3.10

2.72 2.69 2.98 2.17 2.49 2.98 2.13 2.45

miR-147b miR-411(↑) miR-1195 miR-1057 miR-218(↑) miR-130b miR-20a miR-706 miR-486-3p miR-486-5p

2.01 2.09 2.07 2.11 2.01 2.01 2.17 2.06 2.16 2.06

miR-739 miR-187(↑) Let-7e Let-7j(↑) miR-1433

2.03 2.47 2.01 2.09 2.16

a TS-7: tail suspension for 7 days; Value represented downregulated fold change except where indicated up by arrow.

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Fig. 2. Alteration of miRNAs induced by tail suspension (TS). Rats were subjected for 7 and 14 days of TS and qRT-PCR was used to validate the miRNAs expression change presented in microarray results. MiRNAs expression levels were calculated by the 2-ΔΔCt method using snRNA U6 for normalization. Values were presented as mean7 SD (n¼14, nPo 0.05, nnPo 0.01).

revealed that the expression of 787 (373 upregulated and 414 downregulated) and 923 (491 upregulated and 432 downregulated) genes were altered at least 2-fold in TS-7 and TS-14 respectively, which represented 397 (233 upregulated and 164 downregulated) genes of common alteration (Supplemental Table S2). These genes contained 14 out of 267 reported in the previous study [21]. Four myogenic and functional genes which listed in the commonly changed gene were identified by qRT-PCR [29]. As shown in Fig. 3, myh4 (38-fold and 51.6-fold) encoding a myosin class II heavy chain and actn3 (5.1-fold and 13.8-fold) anchoring actin-containing thin muscle fiber filaments were significantly increased in TS-7 and TS-14, which maybe explain fiber type shift under microgravity conditions. A3galt2 involved in the synthesis of the isoglobo-series of glycosphingolipids was markedly downregulated about 2.1fold and 4.4-fold in TS-7 and TS-14 respectively. Acin1 was a component of splicing-dependent multiprotein exon junction complex. Its expression was unchanged in TS-7 but decreased 2.0 folds in TS-14. All these changes were consistent with mRNA genechip results and the muscle mass alteration. 3.4. Target prediction of miRNAs and their integration analysis target genes To match the functions of changed miRNAs and mRNA from microarray analysis, we performed DAVID functional chart analysis of changed genes from microarray. Results

showed GO terms of commonly changed genes mainly involving extracellular region (term 1, 2, 9, 13), skeletal system development (term 4, 5, 7), muscle cell differentiation (term 10) (Fig. 4). Gene annotation of functional clusters between TS-7 and TS-14 was almost the same including skeletal system development, muscle system process and response to hormone stimulus (Supplemental Fig. S1). These functions clusters were corresponding to that of altered miRNAs (Fig. 2) as previous reports, such as miR-214 regulating skeletal and muscle development [30,31], miR320 family responsible for insulin stimuli [32] and miR-4865p inhibited protein degradation [33]. Difference of function clusters between TS-7 and TS-14 included neuron development (cluster 2 of TS-7), cell projection organization (cluster 3 of TS-7), actin binding (cluster 1 of TS-14) and so on (Supplemental Fig. S1), suggesting cellular process alteration from quick response to structural adaptation. In general, alteration of mRNA, which function matched that of altered miRNAs, contributed to muscle atrophy. Next, the analysis focused on the miRNA target genes that were commonly predicted by Miranda, PicTar and TargetScanWorm. GO analysis was performed and followed by the function correlation analysis between miRNAs and mRNAs. We searched down-regulated miRNAs and corresponding up-regulated mRNA targets between putative pairs of miRNA and mRNA in the different conditions. There were 11 significant anti-correlated target genes for 4 altered miRNAs (Table 2). Molecular function showed these genes could

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Fig. 3. Effect of tail suspension (TS) on expression of mRNA related to muscle atrophy. QRT-PCR was used to validate mRNA microarray results (myh4, actn3, a3galt2 and acin1) under TS condition. Expression levels were calculated by the 2-ΔΔCt method using GAPDH for normalization. Values were presented as mean 7S.D. (n¼6, nPo 0.05, nnP o0.01).

Fig. 4. Functional Chart analysis of SMG-responsive mRNA. Common changed genes were loaded into DAVID for Functional Chart analysis. Terms involving were ranked based on significant p value. Bars represent the inverse log of the p value. Related miRNAs in our study were shown in the right.

contribute to the muscle atrophy. Notably, upregulation of Fbxo32, putative target of let-7e which decreased in TS-7, may contribute to the muscle mass loss. 3.5. Pathway analysis of altered miRNAs during TS To explore the importance of altered miRNAs in TS-induced muscle atrophy, KEGG pathway analysis of the putative targets of seven changed miRNAs was performed using DAVID tools. The results showed the cellular pathways regulated by predicted targets included MAPK and Wnt signaling (Fig. 5), which were related to muscle regeneration [34,35]. Interesting was that MAPK signaling pathway was the top in the KEGG pathway analysis of both commonly changed mRNA and putative targets of altered miRNAs responding to TS. Additionally, the expressions of three predicted target genes were examined by qRT-PCR. As shown in Fig. 6, SFRP2, which may be involved in satellite cell activation [36], significantly decreased about 2.1-fold and 3.5-fold in

TS-7 and TS-14 respectively. Another two genes which highlighted in the predicted genes and related with IGF-1 signaling [37–40] were also identified. Results showed that whyz (-2.0-fold and -4.4-fold), target of miR-320 family, and ywhaz (-1.5-fold and -2-fold), target of miR-214, decreased in TS-7 and TS-14 respectively (Fig. 6). 4. Discussion Microgravity induces muscle atrophy, including weight mass decrease and fiber type shift, but the mechanism is far away understood. The major objective of this study was to determine the alteration of miRNAs and mRNAs profile during muscle atrophy induced by TS to simulate microgravity, and especially to confirm their relation with muscle atrophy. Muscle atrophy is directly correlated with diverse pathways which crosstalk and modulate one another at different levels, coordinating protein synthesis and degradation simultaneously. Most of these interact with miRNAs [35,41,42]. So

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Table 2 Anti-correlated genes of differentially expressed miRNAs in TS VS. CN of rat soleus. Gene symbol let-7e abcb9 Tmod2 Fbxo32 Kcnc1 Ptprd miR-214 Chmp4b Dock9 Ptprd miR-221 Ddit4 miR-320 Arid5b Sema6d

GO terms

ATP binding Actin binding Ubiquitin-protein ligase activity Potassium channel activity Protein tyrosine phosphatase activity, cell Adhesion molecule binding Protein transport Regulation of Rho GTPase activity Protein tyrosine phosphatase activity, cell Adhesion molecule binding 14-3-3 protein binding DNA binding, transcription coactivator activity Receptor activity

TS7

TS14

 1.76

 2.01 2.97

2.42 2.40 2.87  1.97 2.69

 1.71 3.75  2.28

10.60 2.03  1.65 3.93 2.26 2.03  1.39 2.74  2.72 2.04 2.14

Fig. 5. Altered cellular pathways under simulated microgravity (SMG). Seven SMG-responsive miRNAs were loaded into TargetScan and DAVID. MiR-4865p and miR-674 were not included in the TargetScan database. The altered cellular pathway was ranked based upon significant p value. Bars represent the inverse log of the p value.

Fig. 6. Effect of tail suspension on expression of mRNA related to muscle atrophy. QRT-PCR was used to examine expression of predicted common targeted gene of altered miRNAs (SFRP2, whyz and ywhaz) under TS condition. Expression levels were calculated by the 2-ΔΔCt method using GAPDH for normalization. Values were presented as mean7 S.D. (n ¼6, nP o 0.05, nnPo 0.01).

pathway analysis of altered miRNAs was performed to further confirm potential function of those miRNAs. Muscle mass decreased quickly in the first week then declined slowly (Fig. 1), which was in accordance with previous reports that up to 37% muscle mass reduction was observed in

the first week of space-flown rat [43] and that muscle atrophy after 91 days’ spaceflight was only slightly greater than that observed after 20 days’ spaceflight, which suggested that an equilibrium may be reached already after two weeks of spaceflight [44]. However, comparison among relative muscle

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mass of CN-7, CN-14 and CN-28 showed a slight decrease of muscle relative mass without statistical difference, which resulted possibly from lonely and decrease of exercise. The primary objective of this studywas to identify the miRNAs expression pattern in the rat soleus muscle under SMG. Previous study revealed 151 miRNAs expressed in the rat soleus muscle and 18 miRNAs were significantly changed after two and/or seven days of TS without change of miR-206 [25]. In present study, we found not only 23 of 174 miRNAs, but also 1313 mRNAs changed at least 2-fold in TS7 and/or TS-14. The expression change of some miRNAs and mRNAs was identified by qRT-PCR. Almost all results of qRT-PCR were consistent with those of array analysis. In accordance with previous report [25], musclespecific miRNAs (miR-1, miR-133, miR-206) expressed abundantly but equally changed little in TS. miR-206 only found in skeletal muscle [13] played an important role in gene expression [45], signaling pathway [46] and regulation of muscle disease [47,48]. It suggested that not all miRNAs responded to TS even if they played important role in the muscle. So it became necessary to determine the miRNAs which respond to TS. However, Allen reported that expression of miR-206 decreased by 50% in gastrocnemius with 12 days spaceflight [21]. The discrepancy maybe result from difference of muscle type. Rodent muscles that express predominantly slow motor units (such as soleus) appear to be more sensitive to unloading stimuli compared to muscles expressing primarily the fast motor units [26]. SFRP2, predicted target gene of miR-206, had a potential role in satellite cell activation [36] and anti-apoptosis effect in hypertrophic scars [40]. Interesting, SFRP2 showed significant decrease in TS-7 and TS-14 which hinted that microgravity inhibited the activation of satellite cells and that other factors influenced SFRP2 expression during microgravity. Alteration of miRNAs showed three patterns: continuous changes during TS (miR-214, miR-486-5p, miR-221), changes at early stage of TS-7 (miR-674 and Let-7e) and at late stage of TS-14 (miR-320b and miR-187). For the continuously changed miRNAs, previous reports have demonstrated that they had important positive effects on anabolism of muscle. miR-214 promoted myogenic proliferation and differentiation [30,31] and miR-486-5p inhibited protein degradation [33]. In DMD, miR-486 (now named miR-486-5p) was identified as regulator of muscle regeneration [47]. It was reported that miR-486 promoted myoblast cell proliferation through phosphatase and tensin homolog deleted on chromosome 10/AKT (PTEN/AKT) pathway [49]. MiR-486-5p significantly declined in the first week of TS which implied an increase of PTEN and decrease of Akt signaling slightly similar to previous report [50]. MiR-221 was able to modulate differentiation and maturation of skeletal muscle cell [27]. The change trend of miR-221 was the same as previous report [25]. Thus, their continuous decrease in present study maybe result in muscle atrophy. Functions of miR-674 were reported little. Targeted genes of Let-7 family involved cell cycle [51], apoptosis [52] and TGF-beta signaling [42]. Moreover, miR320 family which targeted IGF-1 and IGF-1R [32,54] was downregulated in TS-14. Decrease of catabiosis may be related to the decrease of muscle atrophy. So the three

patterns of alteration of miRNAs suggested some cues of muscle atrophy. Combined with their function, changes of mRNAs were also consistent with the progress of muscle atrophy induced by SMG. Three predicted target genes from changed miRNA (Fig. 6) related to IGF-1 signaling showed to decrease after tail suspension by qRT-PCR. Whyz encoded protein 14-3-3 inhibiting protein degradation through binding to IRS1 [37] and IRS2 [39]. Ddit4, regulating 14-3-3 protein binding, was negatively regulated by miR-221 which decreased in mRNA microarray. Ywhaz, encoding 14-3-3zeta, activated PI3K through binding the p85 regulatory subunit [38]. The progression of disuse atrophy involved inhibition of PI3K/Akt [53]. In summary, the decrease of miRNAs (miR-214, miR-486-5p and Let-7e) and genes (whyz and ywhaz) might contribute to muscle atrophy. Decreases of all these anabolic factors response to TS in rat soleus muscle suggested that these miRNAs and mRNAs probably contributed to muscle atrophy progress during tail suspension. GO analysis of commonly changed genes (Fig. 4) and pathway analysis of seven changed miRNAs (Fig. 5) further confirmed that the function of changed miRNAs and mRNAs were related to the muscle atrophy. Fig. 4 showed the first 13 GO terms of commonly changed genes including skeletal system development (term 4, 5, 7), muscle cell differentiation (term 10) (Fig. 4). Some terms showed to directly relate to muscle development (term 10) which suggested that some changed mRNAs regulated muscle atrophy. Some terms matched the functions of changed miRNAs (Fig. 2) according to previous reports, such as miR214 regulating skeletal and muscle development [30,31], miR-320 family responsible for insulin stimuli which is important to muscle function [32]. These results suggested the changed miRNAs and mRNAs were related. Fig. 5 showed that the cellular pathways regulated by predicted targets included MAPK and Wnt signaling, which were related to muscle regeneration [34,35]. So GO and pathway analysis showed the functions of altered genes correlated with the muscle atrophy according to previous reports. These results highlighted the importance of changed mRNAs and miRNAs in muscle atrophy induced by tail suspension. Besides the functional correlation analysis, the analysis of direct regulation between changed miRNAs and mRNAs was a strong evidence about credibility and interaction with muscle atrophy. MiRNAs work through inhibiting the translation of mRNA or degrading it. In present study, we only paid attention to the changed mRNAs that more likely to have potential function related with muscle atrophy. Whether unchanged mRNAs have real function needs to be confirmed by proteomics experiments, that’s the content of following study. SMG-induced muscle atrophy was often accompanied by a phenotypic transformation characterized by a slow- to fast-twitch shift in fiber type [55]. The transformation was partly brought by an expression alteration of MHC isoforms [29]. Inhibition of miR-214 resulted in a reduction or loss of slow-muscle cell types regulated by Hedgehog signaling [56]. In this study, the decrease of miR-214 and increase of myh1 and myh4 provided a cue for muscle mass loss and transformation of muscle fiber type.

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5. Conclusions In summary, this report indicated the expression alteration of miRNAs and mRNAs in rat soleus under TS simulated microgravity conditions. Several miRNAs (miR-214, miR-320, miR-486-5p, Let-7 family) and genes (whyz, ywhaz, SFRP2, myh4 and actn3) responded to TS and accompanied with muscle mass loss, which may involve IGF-1 signaling, PI3K/ PTEN/AKT pathway. The altered model of these miRNAs and mRNA may explain the trend of muscle mass loss. Detail roles of these miRNAs and genes in muscle atrophy need to be further investigated.

Acknowledgments The manuscript was written through contributions of all authors. We thank Jianghui Xiong, Fengji Liang and Xu Li for helpful discussion while microarray result analysis. This work was supported by grants from the National Basic Research Program of China (973 Program No. 2011CB707704); National instrumentation program of China (NO. 2013YQ190467), Advanced space medico-engineering research project of China (2012SY54A1602), the State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center (SMFA11K01, SMFA12B02). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j. actaastro.2014.11.021.

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[53] P. Zhang, X. Chen, M. Fan, Signaling mechanisms involved in disuse muscle atrophy, Med. Hypotheses 69 (2007) 310–321. [54] X.P. Ren, J. Wu, X. Wang, M.A. Sartor, J. Qian, K. Jones, P. Nicolaou, T.J. Pritchard, G.C. Fan, MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20, Circulation 119 (2009) 2357–2366. [55] K.M. Baldwin, F. Haddad, Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms, Am. J. Phys. Med. Rehab 81 (2002) S40–51. [56] A.S. Flynt, N. Li, E.J. Thatcher, L. Solnica-Krezel, J.G. Patton, Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate, Nat. Genet. 39 (2007) 259–263. Hongjie Xu is carrying out successive postgraduate and doctoral programs of study in school of life sciences, northwestern polytechnical university, Xi’an of China. He got bachelor’s degree in 2009 as Excellent graduates. Currently, he studies in China Astronaut Research and Training Center. His research focuses on the microgravityinduced bone loss and muscle atrophy.

Feng Wu, Research Assistant, state key laboratory of space medicine fundamentals and application, China astronaut research and training center. Research Interests: The structure and function changes of different cells exposed to microgravity conditions; Methylation and gene expression regulation under microgravity condition; Prevention of microgravity-induced bone loss and countermeasures for Spaceflight.

Hongqing Cao is the Senior Laboratory Technician at animal experimental center, state key laboratory of space medicine fundamentals and application, China astronaut research and training center. Her research interest is the experimental animal science. She is expert in several animal model, including the rodent hindlimb suspension and primate species bed-rest. She is responsible for the order, feeding and dissection of all experimental animal in the lab.

Guanghan Kan is the director of animal experimental center, state key laboratory of space medicine fundamentals and application, China astronaut research and training center. His research interest is the experimental animal science. He is expert in several animal model, including the rodent hindlimb suspension and primate species bed-rest. He is responsible for the management of all animal experiments in the lab.

Hongyu Zhang, PhD, Research Assistant, state key laboratory of space medicine fundamentals and application, China astronaut research and training center. Research Interests: The structure and function changes of different cells exposed to microgravity conditions; Signaling and gene expression regulation under microgravity condition; the role of actin cytoskeleton in the signaling pathway induced by microgravity; Prevention of microgravity-induced diseases and countermeasures for Spaceflight.

H. Xu et al. / Acta Astronautica 107 (2015) 40–49 Ella W. Yeung is an appointed member of the Physiotherapists Board of Hong Kong. Ella serves as sports physiotherapist in many international sporting events and served many National team players for injury prevention and sports rehabilitation. Ella's research focuses on the mechanisms underlying skeletal muscle function under physiological and pathological conditions. She has published in many international journals such as Journal of Physiology, Journal of Applied Physiology and British Journal of Sports Medicine. Peng Shang is the president of school of life sciences at northwestern polytechnical university, director of key laboratory for space bioscience and biotechnology. His research interests include the field of space biology and space medicine. Space bone loss, response of bone to microgravity and other mechanical stress, biology effect of magnetic fields.

49 Zhongquan Dai, PhD, associate professor, state key laboratory of space medicine fundamentals and application, China astronaut research and training center. Research Interests: The structure and function changes of different cells exposed to microgravity conditions; signaling and gene expression regulation under microgravity condition; the role of actin cytoskeleton in the signaling pathway induced by microgravity; prevention of microgravity-induced diseases and countermeasures for spaceflight. Yinghui Li is director of state key laboratory of space medicine fundamentals and application, vice chief designer at China astronaut research and training center, Member of International Academy of Astronautics, committee member of COSPAR China, In the China manned spaceflight program, she is responsible for aerospace medicine discipline and related space biological research in series of Shenzhou flights. Her research interests include the effects and mechanisms of microgravity environment on physiological system and cellular & molecular biology, especially bone loss, cardiovascular remodeling, etcetera.