| Objective: To investigate whether Guanosine triposphate cyclohydrolase 1(GCH1)participates in the inflammatory response of microglia by regulating lncRNAs,and for the first time on the long non-coding RNA of mouse microglia with knockdown of GCH1(Long noncoding RNA,lncRNA)and mRNA expression profile for high-throughput sequencing analysis.Through bioinformatics screening and verification of inflammation-related lncRNAs regulated by GCH1,to further explore the molecular mechanism of GCH1’s role in neuropathic pain.Methods: The BV2 cell line of mouse microglia with good growth status was selected,and the expression of GCH1 gene in mouse microglia was knocked down by the method of transfection with virus vector.The BV2 cell under different intervention conditions were divided into two groups: Ad-NC group(control group),Ad-shCH1 group(experimental group).Then the Ad-shCH1 and Ad-NC two sets of samples were analyzed by the second-generation high-throughput sequencing(Kangcheng Biotechnology Co.,Ltd.,Shanghai),and the differentially expressed lncRNAs and mRNAs were screened.Secondly,using Gene Ontology(GO)and KEGG database(Kyoto Encyclopedia of Genes and Genomes,KEGG)approach analysis to study the biological functions of differentially expressed mRNA in the sequencing data.Co-expression related data analysis of differentially expressed lncRNAs and mRNAs related to inflammation was carried out,and finally the selected lncRNAs and mRNAs were verified by real-time quantitative PCR.Results: 1.High-throughput sequencing showed that a total of 6619 lncRNAs were detected in the Ad-shCH1 group(experimental group)and Ad-NC group(control group).Compared with the Ad-NC group,47up-regulated and 27 down-regulated lncRNAs were found in the Ad-shCH1 experimental group(P <0.05).At the same time,we detected 22,499 mRNAs in the sequencing data.Among these mRNAs,compared with the control group,there were 155 significantly up-regulated and 168 down-regulated mRNAs respectively(P<0.05).2.Compared with the control group,in terms of biological processes,the most significant GO analysis in the experimental group was tetrahydrobiopterin biosynthetic process(GO:0006729),and the most significant up-regulation was myelin assembly(GO:0032288);in terms of cellular components,In the experimental group,the most down-regulated GO analysis was intracellular membrane-bounded organelle(GO:0043231),and the most up-regulated GO analysis was compact myelin(GO:0043218);in terms of molecular function,the most down-regulated GO analysis in the experimental group was mitogen-activated protein kinase(GO:0051019),the most obvious increase is Transferase activity(GO:0016740).KEGG pathway analysis is a functional analysis of genes.In our study,only one down-regulated pathway was analyzed: but it was related to the folic acid synthesis pathway;no up-regulated pathway was analyzed.3.Five lncRNAs(ENSMUST00000156079,ENSMUST00000180643,ENSMUST00000205634,ENSMUST00000218450,ENSMUST00000125824)related to mitogen-activated protein kinase related to mitogen-activated protein kinase were screened out through co-expression analysis and GO analysis,and mRNAs related to inflammation were further searched through network analysis.4.We measured the mRNA expression levels of 5 lncRNAs(lncRNAENSMUST00000156079,ENSMUST00000180643,ENSMUST00000205634,ENSMUST00000218450,ENSMUST00000125824)and MAPK14 related to mitogen-activated protein kinase by real-time PCR.The results showed that the relative expression levels of ENSMUST00000156079,ENSMUST00000180643,ENSMUST00000205634,ENSMUST00000218450 and MAPK14 were down-regulated in the experimental group compared with the control group.The expression level of ENSMUST00000125824 is meaningless.Conclusion: It has been agreed that microglia inflammatory response is involved in the occurrence and development of neuropathic pain,and our previous studies have shown that GCH1 promotes microglia inflammatory response and participates in neuropathic pain.In recent years,domestic and foreign studies have found that long non-coding RNAs play an important role in neuropathic pain,but many mechanisms are still unclear.Therefore,this study aims to investigate whether the related lncRNA regulated by GCH1 is involved in the inflammatory response of microglia.In this study,after sequencing the knockdown GCH1 microglia,GO analysis revealed that GCH1 can positively regulate the mitogen-activated protein kinase inflammatory pathway.Then co-expression analysis to the five lncRNA and mRNA related to this pathway,and through qPCR to verify the differential expression of lncRNA and MAPK14 mRNA expression levels,found that GCH1 can positively regulate inflammation-related lncRNA and mRNA expression levels.This also provides new and relevant ideas for studying the involvement of microglia in neuropathic pain. |
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