| Microgravity is an important contributor to the formation of adverse effects during spaceflight.Caenorhabditis elegans is a classic model animal,and the simulated microgravity analysis system has been established in C.elegans recently.Meanwhile,insulin,Wnt,and p38 mitogen-activated protein kinase(p38 MAPK)signaling pathways have been identified to be required for toxicity induction of simulated microgravity in nematodes.Micro RNAs(miRNAs)are non-coding RNAs with length of approximately 22 nucleotides,and Lnc RNAs are non-coding RNAs with length greater than 200 nucleotides.Both miRNAs and lnc RNAs play important functions in the regulation of gene expression.However,the molecular functions of miRNAs and lnc RNAs in regulating the toxicity of simulated microgravity are still largely unclear in nematodes.This study aimed to identify and to analyze the functions of miRNAs and lnc RNAs in regulating the toxicity of simulated microgravity in nematodes.1.Systematic identification of microRNAs in response to microgravity stress in nematodesAfter treatment in Rotary Cell Culture System(RCCS)at 30 rpm for 24 h,we observed that 19 miRNAs(16 down-regulated and 3 up-regulated)were dysregulated by simulated microgravity.Based on the gene ontology analysis,the possible biological processes mediated by the candidate miRNA were classified into the categories including development,reproduction,cellular localization,etc.Based on the KEGG analysis,60 signaling pathways were identified for the candidate miRNAs in simulated microgravity treated nematodes.We then investigated the toxicity of simulated microgravity in 8 available genetic miRNA mutants(mir-51,mir-52,mir-67,mir-77,mir-78,mir-85,mir-252,and let-7).Mutation of let-7,mir-67,mir-85 or mir-252 induced a resistance to the toxicity of simulated microgravity.Using q RT-PCR technique,we observed that the simulated microgravity could significantly decrease the expressions of let-7,mir-67,mir-85,or mir-252.These results suggested that alteration in the expressions of let-7,mir-67,mir-85 and mir-252 mediated a protective response to simulated microgravity.2.Intestinal long non-coding RNAs in response to simulated microgravity stress in nematodesLong non-coding RNAs(lnc RNAs)are important response molecules to environmental stresses in organisms.We further employed the animal model of C.elegans to examine the functions of intestinal lnc RNAs in regulating the response to simulated microgravity stress.Among the intestinal lnc RNAs,linc-2,linc-46,linc-61,and linc-78 were increased by simulated microgravity,and linc-13,linc-14,linc-50,and linc-125 were decreased by simulated microgravity.Among these 8 intestinal lnc RNAs,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 of DAF-16,SKN-1,and HLH-30.RNAi knockdown of daf-16,skn-1,or hlh-30 could suppress the resistance of linc-50(RNAi)nematodes to the toxicity in simulated microgravity treated nematodes.Therefore,intestinal linc-50 can regulate the response to simulated microgravity by suppressing the functions of downstream DAF-16-medaied insulin signaling,SKN-1-mediated p38 MAPK signaling,and HLH-30-medaited autophagy signaling.In this study,we further found that the intestinal linc-50 could also target to miRNA molecule of let-7 to regulate the toxicity of simulated microgravity.Meanwhile,let-7regulated the toxicity of simulated microgravity by suppressing the functions of its targets of DAF-16 and SKN-1.In intestinal cells,GST-4,GST-5 and GST-7 acted as downstream targets of the SKN-1 to regulate the toxicity of simulated microgravity.Therefore,in simulated microgravity treated nematodes,besides directly targeting to three transcriptional factors(DAF-16,SKN-1,and HLH-30)by intestinal linc-50,a regulation axis of linc-50-let-7-DAF-16/SKN-1 was further identified in the intestine to be required for the response to simulated microgravity. |
