Effect of simulated microgravity and ionizing radiation on expression profiles of miRNA, lncRNA, and mRNA in human lymphoblastoid cells

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Effect of simulated microgravity and ionizing radiation on expression profiles of miRNA, lncRNA, and mRNA in human lymphoblastoid cells Hanjiang Fu , Fei Su , Jie Zhu , Xiaofei Zheng , Changhui Ge PII: DOI: Reference:

S2214-5524(19)30127-0 https://doi.org/10.1016/j.lssr.2019.10.009 LSSR 254

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Life Sciences in Space Research

Received date: Revised date: Accepted date:

28 August 2019 26 September 2019 19 October 2019

Please cite this article as: Hanjiang Fu , Fei Su , Jie Zhu , Xiaofei Zheng , Changhui Ge , Effect of simulated microgravity and ionizing radiation on expression profiles of miRNA, lncRNA, and mRNA in human lymphoblastoid cells, Life Sciences in Space Research (2019), doi: https://doi.org/10.1016/j.lssr.2019.10.009

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Highlights 

Significantly downregulated lncRNAs and mRNAs identified with simulated microgravity



Significantly upregulated lncRNAs and mRNAs identified upon ionizing radiation



Simulated microgravity and radiation additively alter expression patterns of RNAs



Simulated microgravity and radiation synergistically alter expression levels of RNAs

Effect of simulated microgravity and ionizing radiation on expression profiles of miRNA, lncRNA, and mRNA in human lymphoblastoid cells

Hanjiang Fu#, Fei Su#, Jie Zhu, Xiaofei Zheng*, Changhui Ge*

Department of Experimental Hematology and Biochemistry, Beijing Institute of Radiation Medicine, Beijing 100850, China

#

These authors contributed equally to this work.

*

Correspondence:

Changhui Ge, Department of Experimental Hematology and Biochemistry, Beijing Institute of Radiation Medicine, #27 Taiping Rd. Haidian Dist. Beijing, 100850, China.

Phone:

86-10-66931237,

Email:

[email protected];

ORCID:

0000-0002-4215-3828 Xiaofei Zheng, Department of Experimental Hematology and Biochemistry, Beijing Institute of Radiation Medicine, #27 Taiping Rd. Haidian Dist. Beijing, 100850, China.

Phone:

86-10-66932210,

Email:

[email protected].

ORCID:0000-0001-6032-729X

Abbreviations: DAVID, Database for Annotation, Visualization and Integrated Discovery; DDR, DNA damage response; DEGs, differentially-expressed genes; FC, fold-change; GO, gene ontology; IR, ionizing radiation; KEGG, Kyoto Encyclopedia

of Genes and Genomes; lncRNA, long non-coding RNA; MG, microgravity; miRNA, microRNA; PBLs, peripheral blood lymphocytes; PPI, protein–protein interaction; ROS, reactive oxygen species

Abstract In space, multiple unique environmental factors, particularly microgravity and space radiation, pose a constant threat to astronaut health. MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are functional RNAs that play critical roles in regulating multiple cellular processes. To gain insight into the role of non-coding RNAs in response to radiation and microgravity, we analyzed RNA expression profiles in human lymphoblastoid TK6 cells incubated for 24 h under static or rotating conditions to stimulate microgravity in space, after 2-Gy γ-ray irradiation. The expression of 14 lncRNAs and 17 mRNAs (differentially-expressed genes, DEGs) was found to be significantly downregulated under simulated microgravity conditions. In contrast, irradiation upregulated 55 lncRNAs and 56 DEGs, whereas only one lncRNA, but no DEGs, was downregulated. Furthermore, two miRNAs, 70 lncRNAs, and 87 DEGs showed significantly altered expression in response to simulated microgravity after irradiation, and these changes were independently induced by irradiation and simulated microgravity. GO enrichment and KEGG pathway analyses indicated that the associated target genes showed similar patterns to the noncoding RNAs and were suggested to be involved in the immune/inflammatory response including LPS/TLR, TNF, and NF-B signaling pathways. However, synergistic effects on RNA expression and cellular responses were also observed with a

combination of simulated microgravity and irradiation based on microarray and RT-PCR analysis. Together, our results indicate that simulated microgravity and irradiation additively alter expression patterns but synergistically modulate the expression levels of RNAs and their target genes in human lymphoblastoid cells.

Keywords: Long-noncoding RNA; microRNA; simulated microgravity; irradiation; human lymphoblastoid

1. Introduction All living organisms experience multiple physiological changes in response to unique environmental factors, and this is especially true for microgravity (MG) and space radiation. MG reportedly exerts a series of harmful health effects such as bone density loss, muscle loss, metabolic dysfunction (Jonscher et al., 2016), cardiovascular deconditioning, neurovestibular changes (West, 2000; Williams et al., 2009), immune dysfunction with increased opportunistic infections (Gueguinou et al., 2009; Wilson et al., 2007), and DNA damage at the cellular level (Moreno-Villanueva et al., 2017). These health effects have been observed in studies during spaceflight missions or using ground-based simulations of MG (Hargens and Vico, 2016). The influence of MG on the biological effects of space radiation were studied in the 1980s, but conflicting results were reported for various organisms or cells from a limited number of studies. MG was found to influence radiation-induced DNA repair

in some studies (Horneck et al., 1997; Pross et al., 2000), manifesting as an additive effect when these treatments were used in combination. However, in other studies, either synergistic (enhanced) effects (Bucker et al., 1986; Gao et al., 2015) or suppressive effects (Kobayashi et al., 1996) were found. Possible interactions between ionizing radiation (IR) and MG have recently been proposed with respect to the following cellular-responses: damage and signaling by reactive oxygen species (ROS), the DNA damage response (DDR), and expression of genes and proteins (Yatagai et al., 2019). Although unknown, the reason for these controversial results might be due to the difference in biological systems and the various experimental conditions used in these studies (Yatagai et al., 2019). Therefore, it is necessary to understand the molecular and cellular responses to the combined effects of radiation and MG to assess the health risks in future long-term spaceflights such as moon and Mars missions. MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs), functional RNAs that are not translated into proteins, play critical regulatory roles in various pathological and physiological processes. IR induces changes in miRNA expression in vitro and in vivo, according to the cell type, radiation dose, and post-irradiation time (Bugden et al., 2019; Dickey et al., 2011; Girardi et al., 2012; Lu et al., 2016; Simone et al., 2009), and miRNAs regulate the DDR at the transcriptional level (Mao et al., 2014) in a p53-dependent manner (Goeman et al., 2017). Multiple lncRNAs participate in a wide range of cellular processes including p53-dependent DDR (Beer et al., 2017), apoptosis (Su et al., 2018), and immune regulation (Aryankalayil et al.,

2018; Zhou et al., 2019). Recently, some miRNAs expressed under simulated MG conditions

were

reported

to

be

significantly

negatively

correlated

with

irradiation-induced DDR in human peripheral blood lymphocytes (PBLs) (Girardi et al., 2012). However, further studies are still needed to assess whether the simultaneous exposure of cells and organisms to MG and IR produces additive or synergistic consequences, especially based on the genome-wide profiling of non-coding RNAs. Here, we sought to understand the combined effects of simulated MG and IR and to analyze RNA expression profiles in human lymphoblastoid TK6 cells incubated for 24 h under static or rotating conditions to stimulate MG in space, after IR with 2.0 Gy of γ-rays. The functional classification of miRNA- and lncRNA-correlated target genes was then performed and selected RNAs were analyzed by real-time PCR for extended validation.

2. Materials and methods 2.1 Cell culture and irradiation Human lymphoblastoid TK6 cells were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum and maintained in 5% CO2 at 37 °C. The cells were cultured for 24 h under static conditions without (Control) or after irradiation (IR), cultured under rotating conditions for 24 h to simulate MG using Rotary Cell Culture Systems (Synthecon, Houston, TX, US), or cultured under rotating conditions for 24 h after irradiation with γ-rays (MG + IR) (Simons et al.,

2010). The effective gravitational force under simulated MG was reduced to approximately 10−2 g. IR was performed using a

60

Coray source (Beijing Institute

of Radiation Medicine, Beijing, China) with a total dose of 2.0 Gy at a dose rate of 128.67 cGy/min, which is similar to experimental conditions described previously (Mognato and Celotti, 2005).

2.2 RNA extraction and microarray analysis RNA profiling was conducted using the “Human Transcriptome Array 2.0” (Affymetrix, Santa Clara, CA, US), which detects 1232 human miRNAs, 22496 human lncRNAs, and 41807 human mRNAs. Total RNA was extracted from 107 TK6 cells using TRIZOL Reagent (Life technologies, Carlsbad, CA, US), amplified, labeled, and purified using the Ambion WT Expression Kit (Ambion, US) and GeneChip WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA, US) to obtain biotin-labeled cDNA. Array hybridization and washes were performed using the GeneChip® Hybridization, Wash and Stain Kit (Affymetrix, Santa Clara, CA, US). Slides were scanned using the GeneChip® Scanner 3000 (Affymetrix, Santa Clara, CA, US) and Command Console Software 3.1 (Affymetrix, Santa Clara, CA, US) with default settings. Raw data were normalized by the expression console.

2.3 Data processing Data pre-processing was based on single-experiment normalized data using the SBC analysis system (supplied by Shanghai Biotechnology Corporation, Shanghai, China).

The miRNAs, lncRNAs, and differentially-expressed genes (DEGs) with a fold-change (FC) > 2.0 among simulated MG, IR, simulated MG + IR, and control samples were considered significantly different. Overlapping miRNAs, lncRNAs, and DEGs

between

treatment

groups

were

visualized

using

Venny

2.0

(http://bioinfogp.cnb.csic.es/tools/venny/index.html). The gene expression data in this study have been deposited into NCBI’s Gene Expression Omnibus (GEO accession GSE136939).

2.4 Target gene predication and functional classification analysis Target genes regulated by the differentially-expressed miRNAs were identified through a prediction algorithm based on the TargetScan database (Agarwal et al., 2015). The cis-acting target genes of the differentially-expressed lncRNAs were considered to be the genes transcribed within a 10-kb window upstream or downstream of the lncRNAs, whereas the trans-acting target genes were predicated using the RNAplex software (Tafer and Hofacker, 2008). Then, the correlated target genes were obtained by overlapping the miRNA- and lncRNA-target genes with the DEGs using Venny 2.1.0. Functional classification of all DEGs and miRNA- or lncRNA-correlated target DEGs was performed using the Database for Annotation, Visualization and Integrated Discovery online tools (DAVID) (Huang da et al., 2009), based on Gene Ontology (GO) (Harris et al., 2004) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa and Goto, 2000) databases using functional annotation chart options.

2.5 Protein–protein interaction (PPI) network construction The PPI networks of the identified genes and miRNA- or lncRNA-co-related target genes were mapped using STRING v11.0 (https://string-db.org/) to predict protein interactions (Szklarczyk et al., 2019). By integrating these correlations, interaction networks between the target genes and their interactive genes were constructed using Cytoscape software (Version 3.7.1) (Shannon et al., 2003).

2.7 Reverse transcription and real-time PCR Total RNA was isolated from TK6 cells as described previously herein, and cDNA was prepared using ImProm-IITM Reverse Transcriptase (Promega, madison, WI, US) according to the manufacturer’s instructions. Real-time PCR was performed using the Stratagene Mx3000P (Agilent, Santa Clara, CA, US) and TB Green Premix Ex TaqTM kit (Takara, Kusatsu, Japan) according to the kit instructions. The primer sequences are listed in Supplementary Table 1. PCR was performed in triplicate and threshold cycle values of the target genes were normalized to those of the endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) control. Differential expression was calculated according to the 2−ΔΔCT method (Livak and Schmittgen, 2001).

2.8 Statistical analysis Statistical analysis of real-time PCR data was performed using a one-way ANOVA with a Tukey post hoc test with GraphPad Prism 7 software (GraphPad Software, Inc.,

La Jolla, CA). Data are presented as means ± SEM. P < 0.05 was considered significant.

3. Results 3.1 Simulated MG and irradiation induce differential-expression of lncRNAs and genes The lncRNA expression pattern was found to be different between simulated MG and IR conditions (Fig. 1A, Table 1, and Supplementary Table 2). All identified lncRNAs in the simulated MG group were downregulated, and almost all (98.2%) lncRNAs in the IR group were upregulated compared to levels in the control group. Meanwhile, 21.4% of lncRNAs were downregulated and 78.6% of lncRNAs were upregulated in the simulated MG + IR group (Table 1). Interestingly, the differentially-expressed lncRNAs induced by only simulated MG did not overlap with those induced by simulated MG + IR, suggesting that the lncRNAs regulated by simulated MG might not be significantly affected by IR (Fig. 1C). Furthermore, five (35.7%) of 14 lncRNAs in the simulated MG group and 42 (76.4%) of 55 lncRNAs in the IR group were also differentially-expressed in the simulated MG + IR group (Fig. 1C). Taken together, these results suggested that the simulated MG-mediated downregulation of lncRNAs and IR-induced upregulation of lncRNAs additively contribute to the observed effects under combined simulated MG + IR conditions. A similar pattern was observed for mRNAs (i.e. DEGs), with 100% downregulated

DEGs in the simulated MG group and 100% upregulated DEGs in the IR group (Fig. 1B, Table 1, and Supplementary Table 3), with no overlapping DEGs between the simulated MG and IR groups (Fig. 1D). Similar to, but more pronounced compared to that with lncRNAs, 12 (70.6%) of 17 DEGs in the MG group and 46 (82.1%) of 56 DEGs in the IR group were altered in the simulated MG +IR group compared to levels in the control group (Fig. 1D). Details of differential RNA expression between simulated MG and IR groups are presented in Supplementary Figures 1 and 2, showing similar expression levels between simulated MG + IR and IR groups for all upregulated lncRNAs and DEGs, and between simulated MG + IR and simulated MG groups for most downregulated lncRNAs and DEGs. We then performed functional analysis of the DEGs and showed that GO enrichment and KEGG pathway terms were similar between simulated MG + IR and IR groups, but not between these groups and the simulated MG group (Fig. 2, and Supplementary Table 4). This was consistent with data on the expression pattern of lncRNAs and DEGs, and suggested that simulated MG and IR additively regulate cellular responses independently, with the contribution of IR being dominant. However, despite the additive effect on the expression pattern of RNAs under simulated MG and IR conditions, these RNAs could still synergistically contribute to combined cellular responses by regulating separate genes involved in the same signaling network independently. For example, simulated MG downregulated the pro-apoptotic genes BNIP3 and BNIP3L, whereas IR upregulated apoptosis-related genes such as NFKBIA, TNF, and TNFSF10, all of which could affect apoptosis at the

cellular response level (Supplementary Figure 2). Taken together, our results suggested that simulated MG and IR contribute both additively and synergistically to their combined effects through the induction of lncRNA and mRNA expression.

3.2 Differentially-expressed microRNAs and functional analysis of correlated target genes under MG + IR conditions The microarray chip that we used contained 1232 miRNA probes. No miRNAs were differentially-expressed in the simulated MG and IR groups, whereas two miRNAs were differentially-expressed in the simulated MG + IR group compared to levels in the control group. Of these, has-miR-15b was downregulated (FC = 0.48), whereas has-miR-221 was upregulated (FC = 2.48), and both were altered after simulated MG or IR alone (FCs of has-miR-15b were 0.62 and 0.65 and of has-miR-221 were 1.6 and 1.4, for simulated MG and IR, respectively). This might indicate an interactive effect of both radiation and microgravity with a combination of simulated MG and IR. A total of 31 DEGs were found to be associated with the two miRNAs, and GO enrichment and KEGG pathway analyses indicated that these were involved in apoptosis process, the immune response including positive regulation of IL6/12 production, and NF-B, NF, and LPS/TLR signaling pathways (Fig. 3A and B). The PPI network showed that these correlated target DEGs were associated with NF-B/TLR4 and TP53/MDM2 sub-networks, which were affected by both simulated MG (downregulated) and IR (upregulated) (Fig. 3C).

3.3 Functional analysis of lncRNA-associated target genes under simulated MG + IR We next analyzed the lncRNA-target DEGs in response to simulated MG + IR and found 22 DEGs that were correlated with these lncRNAs. GO enhancement and KEGG analyses were performed along with DAVID analysis, which indicated that the lncRNA-associated target DEGs were involved in immune and inflammatory responses including cytokine-cytokine receptor interaction and LPS/TLR, TNF, NF-B, and p53 signaling pathways (Fig. 4). The regulation of these cellular responses showed similar patterns to the expression of lncRNAs between IR and simulated MG + IR groups. PPI analysis of lncRNA-associated target DEGs in the simulated MG + IR group indicated an association with cytokine interaction and inflammation-related regulation networks including the TLR-mediated NF-B pathway and inflammatory response, in which almost all involved DEGs were upregulated (Fig. 4C). Real-time PCR analysis verified the expression of selected lncRNAs and DEGs (Fig. 5) and showed that simulated MG + IR might induce synergistic effects on the expression levels of RNAs. This was consistent with microarray results indicating that most lncRNAs and DEGs did not respond to simulated MG but were altered by the combined action of simulated MG and IR, or were expressed at higher levels in the simulated MG + IR group compared to the effects of simulated MG and IR treatments (Supplementary Files). Interestingly, we found that most

lncRNA-associated target

DEGs

did

not

overlap

with

miRNA-associated target DEGs, except for some key DEGs in common pathways, such as TLR4, TNFRSF9, MDM2, and ZMAT3 (Fig. 3C and 4C).

4. Discussion Previously, concerns were raised regarding the effects of MG on radiobiological processes in space, although conflicting results were reported for various organisms or cells (Yatagai et al., 2019). Here, we screened and examined the expression profiles of miRNAs, lncRNAs, and DEGs in human lymphoblastoid TK6 cells under simulated MG and IR conditions. Our analyses indicated that the profiles of lncRNAs and DEGs induced by simulated MG were unrelated to those induced by IR, indicating that the two factors might exert both additive and synergistic effects on biological processes, with a dominant contribution by IR. The miRNA and mRNA profiles in human PBLs were previously investigated under simulated MG and IR conditions, and the results showed that simulated MG could alter the expression of miRNAs in PBLs compared to that with 1 g incubation (Girardi et al., 2014) or decrease the number of radio-responsive miRNAs and affect the IR-induced DDR in irradiated PBLs (Girardi et al., 2012). However, we did not find similar effects on miRNAs under similar experimental conditions. This disparity might be due to the differences in human PBLs used. In the previous study, freshly isolated PBLs from healthy donors were used, and the effects of simulated MG on radiated PBLs were compared; in contrast, we used cultured human lymphoblastoid TK6 cells for our experiments. Our results are consistent with another report that showed no significant alterations in miRNA expression in lymphoblastoid TK6 cells treated with -irradiation (Marsit et al., 2006). Further, in the study by Girardi et al., GADD45A expression was increased under 2 Gy + 1 g conditions and remained

unchanged under 2 Gy + simulated MG conditions (Girardi et al., 2012). This was inconsistent with the results of a previous study by Wei et al. who showed synergistic effects between IR and simulated MG, with markedly higher expression of GADD45A in self-made human immortal lymphoblastoid cells after incubation in a rotating bioreactor compared to that with static irradiation with a cumulative low dose of γ-irradiation up to a total 1.0 Gy (Wei et al., 2012), although Girardi et al. also reported a synergistic effect. The differences between these studies and ours indicated diverse results based on the effects of a combination of simulated MG and IR, even in similar cell types from different sources under similar experimental conditions. This highlights the complex and uncertain nature of the effects of IR on simulated MG. In our study, although no differentially-expressed miRNAs were identified in human lymphoblastoid TK6 cells under simulated MG or IR conditions, two miRNAs were found to be differentially-expressed under combined simulated MG + IR conditions with an interactive effect. Of the two miRNAs, hsa-miR-15b reportedly plays a role in the positive regulation of osteoblast differentiation (Vimalraj et al., 2014) and negative regulation of osteoblast proliferation (Vimalraj and Selvamurugan, 2015; Vimalraj et al., 2016). The other miRNA, hsa-miR-221, reportedly promotes cell proliferation, migration, and differentiation in osteoblasts (Zhao et al., 2013; Zheng et al., 2018), and is downregulated in osteoporosis (Zhang et al., 2017). MG is the major reason for bone density loss in space, and some studies have identified osteoclasts and their precursors as direct targets of MG (Tamma et al., 2009). Furthermore, space radiation synergistically exacerbates bone density loss (Zhang et

al., 2015). Meanwhile, these two miRNAs were also found to be involved in apoptosis processes, the immune response, IL-6/12 production, and TNF and LPS/TLR signaling pathways, which have been reported to be altered after space flight or under simulated MG conditions (Bakos et al., 2002; Beck et al., 2012; Dang et al., 2014; Smith, 2018; Taylor et al., 2014) and in response to IR (Kiang et al., 2018; Yoshino et al., 2014). Taken together, the altered hsa-miR-15b and hsa-miR-221 levels might be involved in osteoblast development and the immune response under combined simulated MG and IR conditions. Regarding lncRNA and DEG expression profiling, we found that all lncRNAs and DEGs induced by IR did not overlap with those induced by simulated MG. Even in the simulated MG + IR group, the differential expression of RNAs was partially mediated by simulated MG and IR. The expression levels of these lncRNAs and DEGs also showed similar patterns between either IR alone and simulated MG + IR groups or simulated MG and control groups. GO and KEGG pathway analyses did not show any apparent pathway in the simulated MG + IR group that was affected by simulated MG, which might be due to the low numbers of affected lncRNA-associated target DEGs in response to short-term MG treatment. However, the altered expression pattern of lncRNAs and DEGs obviously suggested that simulated MG and IR, when applied in combination, independently and additively exert their biological effects. In contrast, microarray and RT-PCR results showed the synergistic effects of simulated MG and IR at the transcriptional level, which are consistent with previous endpoint studies showing that simulated MG synergistically

affects IR-induced gene expression profiles (Girardi et al., 2012). Furthermore, our data also suggested that RNAs synergistically contribute to combined cellular responses by independently regulating separate genes induced by either simulated MG or IR that are involved in processes such as apoptosis, although the final effects might be dependent on the combined expression levels of these pro- and anti-apoptosis genes. These results are consistent with previous reports demonstrating that simulated MG affects cell survival in response to IR (Canova et al., 2005; Mognato and Celotti, 2005). Taken together, simulated MG and IR might be complicated by the regulation of combined effects in human lymphoblastoid cells through different aspects of RNA expression and corresponding cellular responses. The combination of space environmental conditions during spaceflight reportedly results in immune system dysregulation, with increased levels of NF-κB cytokines like TNFα, IL-6, and IFN (Zhou et al., 2012) or IL-8, IL-1ra, CCL2, CCL4, CXCL5, VEGF, and TPO, in astronauts (Crucian et al., 2014). Our results further indicate that these events might be mainly caused by the accumulation of space radiation, via the upregulation of lncRNAs and related DEGs such as those of the NFKBIA, CCL, and TNF families. However, there are some conflicting results showing that MG can significantly decrease LPS-induced TNFα production in RAW264.7 cells (Wang et al., 2014). Furthermore, although many studies have reported increased sensitivity to radiation and decreased DDR under simulated MG, and others have shown no effects of spaceflight on the capacity of cells to repair artificially-induced DNA damage (Moreno-Villanueva et al., 2017), our results indicated activation of the p53-mediated

pathway in response to IR, including MDM2, ZMAT3, and CD82, but not simulated MG. Therefore, the regulatory mechanism underlying the combined effects of simulated MG and IR still requires further investigation.

5. Conclusion In summary, our data suggest that simulated MG and IR produce additive effects on RNA expression patterns and synergistic effects on RNA expression and cellular responses in human lymphoblastoid cells. These effects are regulated by miRNAs and lncRNAs through their target genes, which are involved in apoptosis and immune and inflammatory responses, among other processes. Our findings provide evidence to understand such combined short-term effects and offer new insight into the complex genetic mechanisms underlying physiological changes that occur in response to weightlessness and space radiation.

Acknowledgments We are grateful to Prof. Changyong Wang (Institute of Military Cognition and Brain Sciences, Academy of Military Medical Sciences, Beijing) for technical support in carrying out simulated microgravity culture experiments. This work was supported by the National Natural Science Foundation of China [grant numbers 91540202, 81773038]. We would also like to thank Editage (www.editage.cn) for English language editing. Declarations of interest None.

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Figure legends Fig. 1. Comparison of lncRNA and mRNA expression profiles between simulated microgravity (MG) and irradiation (IR) conditions. (A and B) Heatmap analysis of differentially-expressed lncRNAs and genes among Control (C), MG, IR, and MG + IR groups. (C and D) Analysis of the numbers of differentially-expressed lncRNAs and mRNAs among MG, IR, and MG + IR groups using Venny 2.1.0. Fold-changes > 2.0 were used to indicate differentially-expressed lncRNAs and genes vs. control levels.

Fig. 2. Analysis of differentially-expressed genes in the simulated microgravity (MG) + irradiation (IR) group. (A) GO enrichment and (B) the KEGG pathway analysis of differentially-expressed genes in microgravity (MG), IR, and MG + IR groups. P < 0.05.

Fig. 3. Analysis of the miRNA-associated target genes in the simulated microgravity (MG) + irradiation (IR) group. (A) Biological process enrichment and (B) KEGG pathway analyses of the miRNA-target differentially-expressed genes (DEGs) under MG + IR conditions. P < 0.05. (C) Protein–protein interaction network of the miRNA-associated target DEGs in response to MG + IR treatment. Pink/green circles indicate up/downregulated DEGs and red/blue rectangles indicate up/downregulated miRNAs.

Black

edges

indicate

protein

–

protein

interactions;

red-arrow/blue-empty-diamond edges indicate that miRNAs up/downregulate the DEGs, respectively.

Fig. 4. Analysis of lncRNA-associated target genes in the simulated microgravity (MG) + irradiation (IR) group. (A) Biological process enrichment and (B) the KEGG pathway analyses of lncRNA-associated target differentially-expressed genes (DEGs) under MG + IR conditions. P < 0.05. (C) Protein–protein interaction network of lncRNA-associated target DEGs in response to simulated MG + IR treatment. Pink/green circles indicate up/downregulated DEGs and red/blue rectangles indicate up/downregulated lncRNAs. Black edges indicate protein–protein interactions and red-arrow/blue-empty-diamond edges indicate lncRNA trans/cis target DEGs.

Fig. 5. Validation of selected differentially-expressed lncRNAs and genes. Total RNA was extracted from TK6 cells and subjected to real-time PCR analysis to verify the expression of the lncRNAs n336098, n337041, n344988, and n345751, as well the differentially-expressed genes CCL3, TNF, TNFRSF19, and TLR4. Con: Control, MG: simulated microgravity, IR: irradiation. Data are presented as mean ± SEM. The experiments were performed in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Table 1. The numbers of differentially-expressed lncRNAs and genes after stimulation with microgravity, irradiation, and a combination thereof. RNAs lncRNAs

DEGs

Condition MG IR MG+IR MG IR MG+IR

Total counts 14 55 70 17 56 87

Upregulated 0 54 55 0 56 69

Downregulated 14 1 15 17 0 18

MG: simulated microgravity, IR: irradiation, DEGs: differentially-expressed genes.