The Mechanism Of MiRNA-214 On Zebrafish Offspring Neurotoxicity Induced After Microcystin-LR Exposure

Background:Microcystin leucine arginine（MC-LR）is a neurotoxic toxin produced by cyanobacteria.Our previous work has demonstrated acute and chronic neurotoxicity of MCLR,with cross-generational neurotoxic effects of MC-LR exposure.Epigenetic markers,including altered DNA methylation,histone modification,and non-coding RNA.However,the role of these markers in MC-LR induced progeny nervous system damage remains unclear.Objective:This study aims to analyze the effects of chronic low-dose MC-LR exposure on the regulation of RNA networks in brain tissues,including m RNA,micro RNA（mi RNA）,coviciously closed circ RNA（circ RNA）and long-chain non-coding RNA（lnc RNA）,by conducting high-throughput sequencing on the brain tissues of parent and offspring zebrafish,and to find out the key ce RNA networks and downstream pathways regulating the nervous system,and the mechanism of the effects of paternal MC-LR exposure on the brain development of offspring zebrafish was explored,providing a theoretical basis for clarifying the neurotoxic mechanism of MC-LR.Methods:Part 1 In high-throughput sequencing1.Fertilized eggs were randomly divided into control group,5 μg/L MC-LR groupand 25 μg/L MC-LR group.After three months of exposure,zebrafish entered the sexual maturity stage,and male fish were selected to mate and lay eggs with normal female fish,producing F1 generation.The F1 generation is normally fed until three months.2.Total RNA was extracted from the brain tissues of male zebrafish of F0 generation and F1 generation,and sent to Majorbio Company for full transcriptome sequencing to screen the differentially expressed m RNAs,mi RNAs,lnc RNAs and circ RNAs.3.GO enrichment analysis and KEGG functional enrichment analysis were performed for differentially expressed m RNAs and the F1 generation ce RNA network was constructed.Part 2 In vivo4.The expression of m RNAs and mi RNAs in the brain tissue of F1 generation male zebrafish were detected by RT-PCR.5.The expression of related proteins in PI3K/AKT/CREB signaling pathway in the brain tissue of male zebrafish of F1 generation was detected by Western blot.Part 3 In vitro6.The expression of Magi3 and mi R-214 in HT-22 cells was detected by RT-PCR.7.After overexpression of mi R-214,the expression of Magi3 gene and protein in HT-22 cells was detected to verify whether mi R-214 regulated the expression of Magi3.8.After overexpression of mi R-214,the expression of related proteins in PI3K/AKT/CREB signaling pathway in HT-22 cells was detected by Western blot to verify whether mi R-214 regulated PI3K/AKT/CREB signaling pathway.ResultsPart 1 Paternal exposure to MC-LR affected the expression of RNAs and signaling pathway in brain tissue of F0 and F1 male zebrafish.（1）MC-LR exposure affects the expression of RNAs in the brain tissues of F0 and F1 generations: Through high-throughput sequencing,a large number of m RNAs,mi RNAs,lnc RNAs and circ RNAs were differentially expressed in the brain tissues of male zebrafish of F0 and F1 generations after exposure to MC-LR,compared with the control group.（2）MC-LR exposure affects the expression of PI3K/AKT signaling pathway related proteins in F1 generation: GO and KEGG enrichment analysis of differentially expressed genes in the brain tissue of male zebrafish of F1 generation showed that the PI3K/AKT signaling pathway was significantly enriched,and Western blot analysis showed that the PI3K/AKT signaling pathway was inhibited in the brain tissue of male zebrafish of F1 generation.Part 2 Role of mi RNA in neurotoxicity induced by MCLR（1）The expression levels of Magi3 was up-regulated and mi R-214 was down-regulated detected by RT-PCR.（2）After overexpression of mi R-214,downstream target gene Magi3 was down-regulated in gene and protein expression levels.（3）The PI3K/AKT/CREB signaling pathway was activated by Western blot analysis after mi R-214 overexpression.ConclusionThe exposure of MC-LR of parental zebrafish will affect the abnormal expression of m RNAs,mi RNAs,lnc RNAs and circ RNAs in the brain tissue of male zebrafish of F1 generation,and regulate the signaling pathways related to neural development through the ce RNA network,thus causing brain nerve damage.In order to explore the specific molecular mechanism of MC-LR causing this change,high-throughput sequencing combined with animal and cell experiments found that:exposure of MC-LR in male zebrafish can cause abnormal regulation of ce RNA network in the brain tissue of F1 generation male zebrafish,in which circ RNA-NC₀07112.7₅8139783₅8162427 can be used as the sponge of mi RNA-214 by targeting mi RNA-214.Mediates the regulatory effect of mi RNA-214 on magi3a and affects the development of brain nerves through the PI3K/AKT/CREB signaling pathway.In conclusion,MC-LR inhibited the PI3K/AKT/CREB signaling pathway through the circ RNA-NC₀07112.7₅8139783₅8162427-mi RNA-214-magi3 a ce RNA network,resulting in brain nerve damage.