Molecular Mechanisms For Response To Nanopolystyrene At Predicted Environmental Concentration In Nematode Caenorhabditis Elegans

Background:Recently,nanoplastic pollution and its potential effects on whole ecology and even human health have gradually attracted increasing attention.As one of the most frequently detected pollutants in the environment,nanopolystyrene is widely used in many fields of our daily life.However,the molecular mechanisms of its toxic effects on organisms at environmental predicted concentration is still largely unclear.Caenorhabditis elegans,one of classical model animals,has been widely applied in determing toxic effects and molecular mechanisms of various environmental toxicants.Therefore,we employed the in vivo assay system of Caenorhabditis elegans to determine underlying mechanisms of MAPKs,miRNAs,and LncRNAs in response to nanopolystyrene exposure at environmental predicted concentration.Methods:In this study,in vivo assay model of C.elegans was used to systematically determine the molecular mechanisms of animals in response to nanopolystyrene exposure at predicted environmental concentration.Intestinal ROS production and locomotion behavior were employed as the main endpoints.Nematodes at L1 were exposed to nanopolystyrene suspension with the addition of OP50 or RNAi bacteria,and the endpoints were evaluated at adult-day 3.The SOLi D and Illumina Hiseq^tm 2000 techniques were used to identify the dysregulated miRNAs and LncRNAs in response to nanopolystyrene exposure.The biological functions of miRNAs and LncRNAs in response to nanopolystyrene exposure were predicted and determined by bioinformatics,molecular,and genetical methods.Results:1.Molecular mechanisms of MAPK signaling pathways in response to nanopolystyrene exposure at predicted environmental concentrationMitogen activated protein kinase(MAPK)signaling cascade is a highly conserved signal from prokaryotic yeast to eukaryotes(such as human),and plays a key role in many physiological processes of organisms,such as development,growth and proliferation,immunity and environmental stress.MAPKs can be divided into three types:p38 MAPK,c-Jun N-terminal kinase(JNK)and extracellular signal regulated kinase(ERK).1.1 Molecular mechanism of p38 MAPK signaling pathway in response to nanopolystyrene exposure at predicted environmental concentrationIn the p38 MAPK signaling pathway,NSY-1(a MAPK kinase(MAPKKK)),SEK-1(a MAPK kinase(MAPKK))and PMK-1(a mitogen-activated protein kinase(MAPK))constitute a classic signaling cascade of NSY-1-SEK-1-PMK-1.In wild-type nematodes,prolonged exposure(from L1-larvae to adult day 3)to nanopolystyrene particles increased the expression of pmk-1 encoding a p38 MAPK.Mutation of pmk-1induces a susceptibility to nanopolystyrene toxicity,suggesting that the p38 MAPK signaling mediates a protective response to nanopolystyrene particles.PMK-1 functioned in the intestine to act upstream of two transcriptional factors(ATF-7 and SKN-1),which could further act upstream of XBP-1,a key regulator of endoplasmic reticulum unfolded protein response(ER UPR),to regulate the response to nanopolystyrene particles.PMK-1,ATF-7,SKN-1,and XBP-1 were all required for the induction of intestinal ER UPR in nematodes exposed to nanopolystyrene particles.Therefore,the intestinal p38 MAPK signaling may mediate a protective response to nanopolystyrene particles by activating XBP-1-mediated ER UPR.1.2 Molecular mechanism of JNK MAPK signaling pathway in response to nanopolystyrene exposure at predicted environmental concentrationThe C.elegans JNK MAPK signaling pathway mainly contains JNK-1,homolog of human JNK,and two MAP kinase kinases(MEK-1 and JKK-1).Exposure to nanopolystyrene in the range of?g/L could increase the expression of genes(jkk-1,mek-1,and jnk-1)encoding JNK MAPK signaling pathway.Meanwhile,RNAi knockdown of any of these genes induced a susceptibility to nanopolystyrene toxicity.In the neurons,SNB-1/synaptobrevin was identified as the downstream target of JNK-1/JNK,suggesting the alteration in neurotransmitter signals in nanopolystyrene-exposed nematodes.In nanopolystyrene-exposed nematodes,JNK-1 modulated TBH-1-mediated octopamine signal and CAT-2-mediated dopamine signal.TBH-1 and CAT-2 further regulated the response to nanopolystyrene by affecting the function of corresponding intestinal octopamine receptors(SER-6 and OCTR-1)and intestinal dopamine receptor(DOP-1).In the intestine,DOP-1 regulated the response to nanopolystyrene by activating the downstream signaling cascade in p38 MAPK signaling pathway.Therefore,our data further highlight the crucial role of neuronal JNK MAPK signaling activated alteration in octopamine and dopamine signals in regulating the response to nanopolystyrene in organisms.1.3 Molecular mechanism of ERK MAPK signaling pathway in response to nanopolystyrene exposure at predicted environmental concentrationIn the core ERK MAPK signaling pathway of C.elegans,lin-45 encodes a Raf,mek-2 encodes a MEK,and mpk-1 encodes a ERK.Nanopolystyrene exposure in the range of?g/L could significantly increase expressions of genes(lin-45,mek-2,and mpk-1)encoding ERK MAPK signaling pathway.Nanopolystyrene at the predicted environmental concentration of 1?g/L could only significantly increase the mpk-1 expression.Meanwhile,RNAi knockdown of any of these genes caused a susceptibility to nanopolystyrene toxicity.ERK/MPK-1 acted in the neurons to regulate the response to nanopolystyrene.Moreover,three genes(ins-4,ins-39,and daf-28)encoding insulin peptides were identified as the downstream targeted genes of neuronal mpk-1 in regulating the response to nanopolystyrene.In nanopolystyrene exposed nematodes,neuronal RNAi knockdown of ins-4,ins-39,or daf-28 decreased expression of intestinal daf-2 encoding insulin receptor and increased expression of intestinal daf-16encoding FOXO transcriptional factor.Therefore,the neuronal ERK MAPK signaling responded to nanopolystyrene by modulating the insulin signaling-mediated communication between neurons and intestine in nematodes.2.Identification and functional analysis of microRNAs in response to nanopolystyrene exposure at predicted environmental concentrationmicroRNAs(miRNAs)usually act post-transcriptionally to suppress the expression of many targeted genes.After exposure from L1-larvae to adult day-3,we found that 7 miRNAs(4 down-regulated(mir-39,mir-76,mir-794,and mir-1830)and 3 up-regulated(mir-35,mir-38,and mir-354))were dysregulated by nanopolystyrene.Expressions of these 7 miRNAs were dose-dependent in nematodes exposed to 1–100?g/L nanopolystyrene.Among these 7 miRNAs,we found that only mir-35,mir-38,mir-76,mir-354,and mir-794 were involved in the regulation of response to nanopolystyrene based on phenotypic analysis of both transgenic strains and mutant nematodes.Overexpression of mir-35,mir-38,or mir-354 induced a resistance to nanopolystyrene toxicity,and overexpression of mir-76 or mir-794 induced a susceptibility to nanopolystyrene toxicity,which suggested that these 5 miRNAs mediated a protective response to nanopolystyrene.Gene ontology and KEGG analysis further implied that mir-35,mir-38,mir-76,mir-354,and mir-794 were associated with various biological processes and signaling pathways.Therefore,our results suggest the crucial role of a certain number of miRNAs in response to nanopolystyrene after long-term and low-dose exposure in organisms.3.Identification and functional analysis of LncRNAs in response to nanopolystyrene exposure at predicted environmental concentrationLncRNAs,defined as noncoding RNAs having at least 200 nucleotides and no protein coding ability,and are involved in the control of various biological processes,including the stress response.Based on Hiseq 2000 sequencing and q RT-PCR confirmation,we identified 37 lnc RNAs(22 down-regulated LncRNAs and 15 up-regulated LncRNAs)in response to nanopolystyrene(1?g/L).Using intestinal reactive oxygen species(ROS)production and locomotion behavior as endpoints,we found that RNAi knockdown of linc-2,linc-9,or linc-61 induced a susceptibility to nanopolystyrene toxicity,and RNAi knockdown of linc-18 or linc-50 induced a resistance to nanopolystyrene toxicity.Meanwhile,nanopolystyrene(1 mg/L)increased expressions of linc-2,linc-9,linc-18,and linc-61 and decreased linc-50 expression,suggesting that these 5 LncRNAs mediated two different responses to nanopolystyrene exposure.Bioinformatical analysis implied that these 5 LncRNAs were associated with multiple biological processes and signaling pathways.Therefore,our results demonstrated the crucial roles of LncRNAs in response to long-term exposure to low-dose nanopolystyrene in organisms.Conclusions:In this study,we found that three MAPK signaling pathways acted in different tissues to regulate the response of C.elegans to nanopolystyrene exposure.In the intestine,p38 MAPK signaling pathway regulated the toxicity of nanopolystyrene by activating ER^UPR response.In the neurons,JNK MAPK signaling pathway regulated the toxicity of nanopolystyrene by modulating octopamine and dopamine signals,and ERK MAPK signaling pathway regulated the toxicity of nanopolystyrene by modulating insulin signaling.After exposure from L1 larvae to adult day 3,5 miRNAs were identified to mediate the protective response of C.elegans to nanopolystyrene exposure.Among 8 known dysregulated LncRNAs,only linc-2,linc-9,linc-18,linc-50,and linc-61 were involved in the regulation of nanopolystyrene toxicity.linc-18 alteration mediated the toxicity induction of nanopolystyrene,whereas linc-2,linc-9,linc-50,and linc-61 alterations mediated a protective response to nanopolystyrene.These identified miRNAs and LncRNAs were associated with many biological processes and signal pathways.Our results will be helpful for understanding the molecular mechanisms of response to nanoplastics at predicted environmental concentration in organisms.