The Role And Mechanism Of Circhomer1 In Regulating Methamphetamine-induced Neuronal Injury

BackgroundMethamphetamine(METH)is recognized as one of the commonly worldwide addictive drugs for years.Abuse of METH with chronic doses causes inevitable damages in the brain.in vitro studies,METH exposure induces irreversible neuronal damage including apoptosis and autophagy.Nevertheless,exact molecular mechanisms of METH-induced neurotoxicity remain largely unknown.As one part of non-coding RNA species,circular RNA(circRNA)contributes to multiple biological functions and molecular processes in disease of nervous system.However,underlying connection between circRNA and METH-induced brain changes is still ambiguous.ObjectiveTo establish profile expression of circRNA in METH-treated primary cortical neurons via high-throughput sequencing;to identify differentially expressed circRNA related to METH neurotoxicity and unveil the role of circRNA in regulating METH-induced lethal process and its downstream gene that contributes to this modulation procedure;to predict the relationship between them and addictive behaviors.Methods(1)Profile expression of circRNA in METH-treated primary cortical neurons:We firstly cultured primary cortical neurons and verified lethal effect induced by METH.Then,we performed high-throughput sequencing to screen differentially expressed circRNA in METH-treated group compared to control group and validated circRNA expression via qRT-PCR.Besides,GO analysis and KEGG analysis were employed and we established heat map and volcano plots for annotation.(2)Function analysis of circHomer1 after METH treatment:Based on sequencing data and bioinformatics analysis,we sorted circHomer1 for further study in HT-22 cell line.Using UCSC website to analyze the biological structure of circHomer1,we next sequenced the qRT-PCR product and confirmed its loop structure.According to its back-splicing site,we designed two small interfering RNA(siRNA)targeting the site for down-regulating circHomer1 expression.Then,we performed flow cytometry and western blot to detect the effect on METH-induced neurotoxicity after knockdown of circHomer1.(3)Function analysis of Bbc3 as one downstream gene of circHomer1:We predicted potential miRNA and gene downstream the circHomer1 via TargetScan and starBase,and identified target gene using western blot and qRT-PCR.Subsequently,we applied CCK-8 and apoptosis staining to detect the effect after silencing Bbc3 expression with two siRNA targeting 3’ UTR of Bbc3.(4)Correlation analysis among circHomer1 and Bbc3 expression with CPP score:We established well-developed addicted models through conditioned place preference(CPP)paradigm and assess the possible correlation among circHomer1,Bbc3 and addictive behaviors.Results(1)Detection of circRNA profile in METH-treated primary cortical neurons:We chose 2mM concentration for METH exposure in primary cultured cortical neurons based on MTT assay.And we got 2458 circRNA derived from exons totally via high-throughput sequencing,in which there are 119 up-regulated circRNA and 44 down-regulated circRNA.Besides,we found circRNAs that we identified distributed mostly less than 2000 nucleotide.According to bioinformatics analysis,The analysis of biological process(BP)terms revealed that ’covalent chromatin modification’ and’negative regulation of synaptic transmission’ were major enriched,while ’nervous system development’ was involved as well.In terms of cellular component(CC),localization of specific circRNAs was found to be related with neuron part primarily.As for molecular function(MF),the most enriched terms were ’histone-lysine N-methyltransferase activity’ and ’metal ion binding’.In KEGG analysis,’morphine addiction’ and ’GABAergic synapse’ were associated with drug addiction.Then,we sorted eight circRNA(Whsc1,Homer1,Lin54,Tlk1,Slc8a1,Gpr137c,Rapgef6 and Ankrd11)for identification,in which we discovered that circHomer1 up-regulated for nearly 1.7 fold in primary cultured cortical neurons after METH treatment compared to control group.(2)Function analysis of circHomer1 after METH treatment:We found circHomer1 up-regulated distinctly in HT-22 cell line,and in NAc,Hp,VTA and pFC of addicted models.Sequencing of qRT-PCR product showed that circHomer1 owned the loop structure and was resistant to RNase R digestion,which demonstrated the stability of circHomer1.We also detected that circHomer1 located mainly in the cytoplasm determined by fluorescence in situ hybridization with circHomer1-specific probe using confocal microscope.We designed two siRNAs(siRNA-1 and siRNA-2)targeting back-splice junction of circHomerl in order to knockdown circHomer1 expression.Results of flow cytometry showed that apoptosis rate were apparently higher after treated with METH and decreased both in groups transfected with siRNA-1 and siRNA-2 compared with si-NC group.And siRNAs silencing circHomer1 effectively reduced the cleaved Caspase3 protein level as well.(3)Function analysis of Bbc3 as one downstream gene of circHomer1:We discovered that METH treatment augmented Bbc3 expression at different concentration for 12h or 24h at mRNA or protein level in HT-22 cells.Collectively,Bbc3 expression was distinctly down-regulated after siRNA-1 or siRNA-2 transfection in advance in the presence of METH on both mRNA expression and protein level.Silencing Bbc3 in advance improved cell viability slightly and reduced PI-positive rate and ROS level in the groups after METH exposure.(4)Correlation analysis among circHomer1 and Bbc3 expression with CPP score:At last,we detected that circHomer1 and circTlk1 were both differentially expressed in Hp,NAc,VTA and pFC of METH-addicted models.And there are highly correlation among circHomer1,Bbc3 and addictive behaviors in addicted mice models,respectively.ConclusionIn this study,we firstly unveiled profile expression of circRNA in METH-treated primary cortical neurons via high-throughput sequencing and identified one novel circRNA,circHomer1.Knockdown of circHomer1 could ameliorate neuronal injury induced by METH through down-regulating Bbc3 expression.Otherwise,there are possible correlation among Bbc3,circHomer1 and addictive behaviors.In summary,circHomer1/Bbc3 possibly modulates METH-induced neurotoxicity.