MicroRNAs-mediated Isoflurane Sensitivity Variation In C.Elegans

Objective:1. Screening isoflurane sensitivity of microRNAs mutants in C. elegans by behavioral assays.2. Construct transcription reporter of microRNAs which have been screened, then observe their spatial and temporal expressions in C. elegans.Methods:1. Behavioral Assay-Radial Dispersion Assay:Coordinate movement was scored by the radial dispersion assay. All assays were performed on well-fed young adult animals (72h post egg delivery) at room temperature (20°). Wide-type N2and mutants were exposed to no isoflurane or different concentrations of isoflurane, respectively, then Radial Dispersal Index was recorded.2. Behavioral Assay-Egg-laying Defective:All assays were performed on well-fed young adult worms. Wide-type N2and mutants(dcr-1and55microRNA mutants) were exposed to no isoflurane or different concentrations of isoflurane, respectively, and then Egg-laying Defective was determined.3. Construct transcription reporter of microRNAs associated with isoflurane sensitivity: VmirRNAy.gfp, then plasmid DNA was microinjected to nematode gonad, the spatial and temporal expressions of microRNAs in C. elegans through confocal laser scanning microscope.Results:1. Compared to wild-type N2, dcr-1、eri-3and6microRNAs(mir-83, mir-240&mir-786, mir-77,mir-257,mir-75,mir-241) mutants showed much more obvious decline in dispersal index and significant disorder of coordinate movement, so these mutants were more sensitive to isoflurane (P<0.05)2. Compared to wild-type N2, C. elegans with dcr-1or6microRNAs(mir-241&mir-84, mir-77, mir-247&mir-797, mir-80, mir-46, nDf63;mir-63) mutation displayed much more obviously egg-laying defective and were more sensitive to isoflurane (P<0.05)3. Characteristics of the spatial and temporal expression:mir-80was expressed in the neurons near pharynx and tail while it can also be found in somatic muscle cells. The expression of mir-84mainly existed in neurons closed to pharyng and cauda. mir-797located in the neurons closed to pharyng and cauda and neurons of central body.Conclusions:1. Isoflurane significantly impacted on coordinate movement of C. elegans by radial dispersion assay, dcr-1, eri-3and microRNAs mutation markedly changed C. elegans sensitivity to isoflurane.2. Mutations in dcr-1and6microRNAs(mir-241&mir-84, mir-77, mir-247&mir-797, mir-80, mir-46, nDf63;mir-63) significantly increased C. elegans sensitivity to isoflurane according to assay of egg-laying defective, which suggested that these genes likely play a role in isoflurane induced anesthesia.3. Characteristics of the spatial and temporal expression:mir-80was expressed in the neurons near pharynx and tail while it can also be found in somatic muscle cells. The expression of mir-84mainly existed in neurons closed to pharyng and cauda. mir-797located in the neurons closed to pharyng and cauda and neurons of central body.