| Objective:This project aims to identify the phenotype of miR-31 transfected neural stem cells through in vitro transfection of small nucleic acid molecules,laying a foundation for bioinformatics analysis of miR-31 regulation of neural development.In addition,mouse model of spinal cord injury was constructed to verify whether small nucleic acid molecules of miR-31 could promote the repair of spinal cord injury,and its repair mechanism was briefly discussed.Then,miR-31 MO zebrafish was constructed based on EGFP:HB9zebrafish to observe the changes in the development of motor neurons after miR-31 knockdown,so as to clarify the specific role of miR-31 in the development of the nervous system.According to the phenotypes related to miR-31 regulation of nervous system development,the downstream target genes of miR-31 were predicted,and GO analysis and KEGG analysis were used to study the enrichment of the downstream target genes of miR-31,so as to analyze the key genes and downstream signaling pathways involved in miR-31 regulation of NSCs.Finally,cell experiments were carried out to verify whether the predicted signaling pathways were correct.Through the above experiments,we hope to further clarify the regulatory effect of miR-31 on the development of the nervous system and related molecular mechanisms.Methods:1.Neural stem cells derived from mouse spinal cord were cultured in vitro and transfected with miR-31 small nucleic acid molecules.Cells in multiple fields were counted under fluorescence microscope to calculate the transfection efficiency of miR-31 small nucleic acid molecules on neural stem cells.Micro RNA was extracted from neural stem cells at 1 and 3 days after transfection,and the expression changes of miR-31 were detected.Then immunofluorescence staining and quantitative PCR were used to detect the differentiation of the cells and observe the phenotypic changes of the cells.The mouse spinal cord injury model was constructed,and miR-31 small nucleic acid molecules were injected into the spinal cord injury site locally.The motor function recovery of the mice was evaluated with BMS score and grid score,and the repair after spinal cord injury was detected by fluorescence quantitative PCR and WB at the molecular level.2.During the one-cell phase of zebrafish embryos,8 ng miR-31 morpholino and 8 ng Standard Control were microinjected to knock out the expression of miR-31 in zebrafish.At26 hpf and 48 hpf,the distribution of motor neurons was observed under a fluorescence microscope after zebrafish was anesthesia,and the number of incomplete neurons was counted.Then,AO staining was used to detect whether the loss of motor neurons was caused by apoptosis,and analyzed apoptotic particles in miR-31 MO group and control group statistically.Fluorescence quantitative PCR was used to detect the expression changes of related genes to analyze the causes of this phenomenon.3.Micro RNA target gene prediction websites micro RNA.org,MIRDB,miRwalk and targetscan were used to find the downstream target genes of miR-31,and cross-compare and predict the downstream target genes of miR-31 in 4 databases.Then GO analysis and KEGG analysis were performed on miR-31 by DAVID to study the enrichment of downstream target genes of miR-31.According to the regulation of miR-31 on neural development and the phenotype of NSCs,NCBI Ami GO2 was used to search for the corresponding genes related to biological function and cross-compare them with the downstream target genes of miR-31 to analyze the key genes and downstream signaling pathways involved in the regulation of NSCs by miR-31.4.Fluorescence quantitative PCR detected the expression changes of genes related to Notch signaling pathway and Wnt signaling pathway in total RNA of zebrafish,and analyzed the specific signaling pathways involved.Neural stem cells derived from spinal cord of mice were cultured in vitro,transfected with miR-31 small nucleic acid molecules,and the expressions of genes related to Notch signaling pathway were detected by fluorescence quantitative PCR.DAPT was used to inhibit the expression of Notch signaling pathway,and miR-31 agomir was added to detect the expression changes of related genes.Results:1.Under fluorescence microscope,it can be observed that the neural stem cells transfected with miR-31 small nucleic acid molecules successfully with emitting red fluorescence and have a high transfection efficiency.The fluorescence quantitative PCR results showed that the expression of intracellular miR-31 was regulated after the regulation of neural stem cells by the small nucleic acid molecule miR-31.Immunofluorescence chemical staining and quantitative PCR showed that after miR-31 overexpression,Nestin expression increased and the number of neural stem cells increased.In vivo experiments have confirmed that local injection of miR-31 small nucleic acid molecules after the establishment of mouse spinal cord injury model can promote the repair of motor function and improve the expression of Nestin in the spinal cord.2.Abnormal development of zebrafish can be seen after miR-31 knockout.Under fluorescence microscope,abnormal motor neuron development was observed after the deletion of miR-31 expression at 26 hpf and 48 hfp.In the control group,motor neurons were seen completely and clearly,while motor neuron axons disappeared in the miR-31 group.miR-31 MO zebrafish loses escape response.AO staining results indicated that many apoptotic cells appeared in the areas where the spinal cord motor neurons of zebrafish disappeared.At 48 hpf,a large number of apoptotic cells also appeared in the tail of zebrafish.At 8 dpf,the incomplete tail of zebrafish can be observed under the bright field.Fluorescence quantitative PCR showed that HB9 expression was down-regulated and apoptosis-related gene expression was up-regulated.3.By cross-comparing target gene prediction websites,387 downstream target genes predicted by miR-31 were obtained.In the biological process in which the downstream target genes of miR-31 were enriched,GO analysis showed that 4 of the first 15 were related to the development of the nervous system.KEGG analysis showed that 2 of the signaling pathways in which the downstream target genes were involved were related to the development of the nervous system.Through comparison with phenotypic related genes,Numb and Axin-1 in the downstream target genes of miR-31 appear repeatedly in relevant biological processes.4.After knocking down the expression of miR-31,the Axin-1 expression of zebrafish was down-regulated and Wnt signaling pathway was activated,while the Numb expression was up-regulated and the Notch signaling pathway was inhibited.After NSCs regulated miR-31 expression in mice,Numb expression was inhibited,Notch signaling pathway was activated,and the reverse situation occurred after miR-31 expression was down-regulated.Notch signaling pathway was inhibited after DAPT treatment of NSCs in mice,and a small amount of recovery was observed after the addition of miR-31.The expression of miR-31 was up-regulated after DAPT treatment of NSCs in mice.The expression of Nestin was decreased after DAPT treatment of NSCs in mice,and the expression of Nestin was recovered after the addition of miR-31.Conclusion:1.Small nucleic acid molecules of miR-31 can be self-transfected into neural stem cells without the help of transfection auxiliary reagent and can regulate the expression of miR-31 in neural stem cells.One end of miR-31 small nucleic acid molecule is connected with Cy3 luminescent group,so it can make cells emit red fluorescence after transfection into cells,which is conducive to the statistics of successful transfection cells.The overexpression of miR-31 nucleic acid molecule promoted the proliferation of NSCs,while the inhibition of miR-31 expression inhibited the proliferation of NSCs.Mir-31 small nucleic acid molecule can promote the endogenous NSCs proliferation after spinal cord injury,thus promoting the repair of motor function.2.miR-31 knockout zebrafish model was constructed by Morpholino.After miR-31 knockout,motor neuron of zebrafish develop abnormally,and zebrafish lost motor function.AO staining showed that there were many apoptotic cells in the central nervous region and tail of miR-31 MO zebrafish.The abnormal development of zebrafish tail was observed in the open field,and the knockdown of miR-31 would lead to a large number of apoptosis of zebrafish stem cells.After miR-31 knockout,the expression of neuron-related genes was down-regulated,and the expression of apoptosis-related genes was up-regulated.3.The downstream target genes of miR-31 are closely related to the development of the nervous system.miR-31 regulates the proliferation of NSCs by regulating Notch signaling pathway or Wnt signaling pathway.4.miR-31 regulates the proliferation of NSCs by regulating Notch signaling pathway.After DAPT treatment of NSCs in mice,the expression of miR-31 could be up-regulated to activate Notch signaling pathway.DAPT can inhibit NSCs,and upregulation of miR-31 expression can relieve the inhibitory state. |
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