Intestinal long non-coding RNAs in response to simulated microgravity stress in Caenorhabditis elegans

Long non-coding RNAs (lncRNAs) are important in regulating the response to environmental stresses in organisms. In this study, we used Caenorhabditis elegans as an animal model to determine the functions of intestinal lncRNAs in regulating response to simulated microgravity stress. Among the intestinal lncRNAs, linc-2, linc-46, linc-61, and linc-78 were increased by simulated microgravity treatment, and linc-13, linc-14, linc-50, and linc-125 were decreased by simulated microgravity treatment. Among these 8 intestinal lncRNAs, RNAi knockdown of linc-2 or linc-61 induced a susceptibility to toxicity of simulated microgravity, whereas RNAi knockdown of linc-13, linc-14, or linc-50 induced a resistance to toxicity of simulated microgravity. In simulated microgravity treated nematodes, linc-50 potentially binds to three transcriptional factors (DAF-16, SKN-1, and HLH-30). RNAi knockdown of daf-16, skn-1, or hlh-30 could suppress resistance of linc-50(RNAi) nematodes to the toxicity of simulated microgravity. Therefore, our results provide an important basis for intestinal lncRNAs, such as the linc-50, in regulating the response to simulated microgravity in nematodes.


Identification of dysregulated genes induced by intestine-specific RNAi knockdown of linc-50
in simulated microgravity treated nematodes. We next focused on the identification of downstream genes of linc-50, one of the candidate lncRNAs whose RNAi knockdown induced a resistance to toxicity of simulated microgravity. Based on HiSeq 2000 sequencing, 43 genes were dysregulated by intestine-specific RNAi knockdown of linc-50 in nematodes treated with simulated microgravity for 24 h ( Fig. 4a; Table S7). Meanwhile, expressions of these 43 genes were also affected by simulated microgravity treatment (

Potential transcriptional factors targeted by intestinal linc-50 in simulated microgravity treated nematodes.
During the control of biological processes, lncRNAs can act as 'ligands' of transcriptional factors 33 . In nematodes, the candidate transcriptional factors targeted by lncRNAs have been identified by ChIP-SEQ technique to determine transcriptional factors binding site regions of lncRNAs, together with antibodies against transcriptional factors and immunoprecipitate nucleic acids 34,35 . Among the dysregulated genes induced by simulated microgravity treatment, 3 genes (daf-16, skn-1, and hlh-30) encode transcriptional factors potentially targeted by linc-50 (Fig. 5a). The treatment with simulated microgravity could significantly increase expressions of daf-16, skn-1, and hlh-30 ( Fig. 5b; Table S10). Simulated microgravity treatment further increased both expression and nucleus localization of DAF-16::GFP in intestinal cells ( Fig. S4a; Table S11). Similarly, the simulated microgravity also increased both expression and nucleus localization of SKN-1::GFP in intestinal cells ( Fig. S4b; Table S11). Moreover, in simulated microgravity treated nematodes, intestine-specific RNAi knockdown of linc-50 significantly increased expressions of daf-16, skn-1, and hlh-30 ( Fig. 5c; Table S10). Additionally, after simulated microgravity treatment, both expression and nucleus localization of DAF-16::GFP were increased by RNAi knockdown of linc-50 ( Fig. S4; Table S11). These data implied that DAF-16, SKN-1, and HLH-30 might function as the targeted transcriptional factors by intestinal linc-50 in simulated microgravity treated nematodes. DAF-16 is a FOXO transcriptional factor in insulin signaling pathway involved in the regulation of response to simulated microgravity 21 . SKN-1 is a Nrf protein in p38 MAPK signaling pathway, and was required for the regulation of response to simulated microgravity 23 . HLH-30 is a transcriptional factor governing the autophagy induction, and required for the regulation of stress response 22 . PRIdictor (protein-RNA interaction predictor) is a tool to predict mutual binding sites in RNA and protein at nucleotide or residue level (http://bclab .inha.ac.kr/pridi ctor). PRIdictor was further used to predict binding sites between linc-50 and DAF-16, SKN-1, or HLH-30. The DAF-16 protein contains 25 possible nucleotide binding sites for linc-50 (Fig. S5a), and the linc-50 contains 40 possible residue binding sites for DAF-16 (Fig. S5b). The SKN-1 protein contains 25 possible nucleotide binding sites for linc-50 (Fig. S5c), and the linc-50 contains 82 www.nature.com/scientificreports/ possible residue binding sites for SKN-1 (Fig. S5d). The HLH-30 protein contains 2 possible nucleotide binding sites for linc-50 (Fig. S5e), and the linc-50 contains 5 possible residue binding sites for HLH-30 (Fig. S5f).

Genetic interaction among DAF-16, SKN-1, and HLH-30 in the intestine to regulate response to simulated microgravity. After simulated microgravity treatment, genetic interaction analysis indicated
that double intestine-specific RNAi knockdown of daf-16 and skn-1 could cause the more severe ROS production than that in daf-16(RNAi) or skn-1(RNAi) nematodes ( Fig. 7a; Table S15), suggesting that DAF-16 and SKN-1 acted in parallel pathways to regulate response to simulated microgravity. Similarly, after simulated microgravity treatment, double intestine-specific RNAi knockdown of daf-16 and hlh-30 could cause the more severe ROS production than that in daf-16(RNAi) or hlh-30(RNAi) nematodes ( Fig. 7a; Table S15), suggesting that DAF-16 and HLH-30 also acted in parallel pathways to regulate response to simulated microgravity. Moreover, double intestine-specific RNAi knockdown of skn-1 and hlh-30 led to the more severe ROS production in simulated microgravity treated nematodes than that in simulated microgravity treated skn-1(RNAi) or hlh-30(RNAi) nematodes ( Fig. 7a; Table S15), suggesting that SKN-1 and HLH-30 further acted in parallel pathways to regulate response to simulated microgravity. Data on efficiency for intestinal RNAi knockdown of daf-16, skn-1, or hlh-30 is shown in Figs. S6, S7 and Tables S13, S14.

Discussion
Our previous studies have implied the crucial role of intestinal barrier in response to simulated microgravity in nematodes 19,21,[23][24][25] . Firstly, simulated microgravity could activate the protective responses (such as mt UPR) in intestinal cells 21 . Secondly, simulated microgravity could cause damage on intestinal barrier, such as the enhanced intestinal permeability 19 . Thirdly, some important signaling pathways (such as insulin, p38 MAPK, www.nature.com/scientificreports/ and Wnt signaling pathways) could be activated in the intestine, and were involved in the regulation of response to simulated microgravity [23][24][25] . Thus, in this study, we tried to identify the intestinal lncRNAs involved in the regulation of response to simulated microgravity in nematodes. Among intestinal lncRNAs, simulated microgravity treatment for 8 or 24 h could increase expressions of linc-2, linc-46, linc-61, and linc-78, and decrease expressions of linc-13, linc-14, linc-50, and linc-125 (Fig. 2; Table S2). Previous study has also suggested that exposure to 1 mg/L graphene oxide (GO) could decrease expression of linc-14 28 . Additionally, exposure to 1-100 μg/L nanopolystyrene (100 nm) could also increase expressions of linc-2 and linc-61, and decrease linc-50 expression 29 . These results implied the possible conserved property of linc-2, linc-14, linc-50, and linc-61 in response to environmental toxicants or stresses.
Among the 8 dysregulated intestinal lncRNAs in simulated microgravity treated nematodes, we further observed the susceptibility of linc-2(RNAi) and linc-61(RNAi) nematodes to toxicity of simulated microgravity and the resistance of linc-13(RNAi), linc-14(RNAi), and linc-50(RNAi) nematodes to toxicity of simulated microgravity ( Fig. S1; Table S3). These observations suggested that the alteration in expressions of linc-2, linc-13, linc-14, linc-50, and linc-61 mediated a protective response to simulated microgravity. The resistance to toxicity of GO could also be detected in linc-14(RNAi) nematodes 28 . Additionally, RNAi knockdown of linc-2 or linc-61 could result in the susceptibility to toxicity of nanopolystyrene, and RNAi knockdown of linc-50 caused the resistance to toxicity of nanopolystyrene 29 .
Moreover, our results demonstrated the important functions of linc-2, linc-13, linc-14, linc-50, and linc-61 in the intestine to regulate response to simulated microgravity. Nevertheless, besides the expression in the intestine, linc-2, linc-13, linc-50, and linc-61 can also be expressed in other tissues in nematodes 31 . Thus, we did not exclude the possibility that these 5 candidate lncRNAs can also act in other tissues to regulate the response to simulated microgravity.
In conclusion, we used C. elegans as an animal model to determine the functions of intestinal lncRNAs in regulating response to simulated microgravity stress. We identified 5 intestinal lincRNAs with protective functions during the control of response to simulated microgravity. Moreover, we found that intestinal linc-50 could regulate response to simulated microgravity by suppressing functions of downstream three transcriptional factors (DAF-16, SKN-1, and HLH-30). Our study provides an important molecular basis for lncRNAs in the intestine to regulate response to environmental exposures in nematodes.
Toxicity assessment. Considering the sensitivity in assessing toxicity of simulated microgravity 18,23,24 , locomotion behavior and ROS production were used as toxicity assessment endpoints.
Locomotion behavior reflects functional state of motor neurons, and was evaluated by head thrash and body bend 38 . After the treatment, nematodes were washed using M9 buffer first. The nematodes were allowed for a 1-min recovery on surface of NMG plate. Under a dissecting microscopy, a head thrash is defined as an alteration in direction of posterior bulb part, and the head thrash frequency was recorded during the duration of 1 min. A body bend is defined as an alteration in direction of bending at the middle body, and the body bend frequency was recorded during the duration of 20 s. Forty nematodes were analyzed per treatment. Three replicates were performed.
ROS production was used to assess activation of oxidative stress in simulated microgravity treated nematodes 39,40 . After the treatment, nematodes were labeled with 1 µM CM-H 2 DCFDA for 3-h without light.