The Role And Mechanism Of LncRNA-Malat1 And Related Transcription Factors Involved In Regulating The Transdifferentiation Of DRG Satellite Glial Cells

Objective:Studies have shown that there is a phenomenon of neurogenesis in the Dorsal root ganglion(DRG)of the peripheral nervous system.This phenomenon is related to a special type of glial cells around the sensory neuron-Satellite glial cells(Satellite glial cells,SGCs).Earlier studies found that SGCs have plasticity and multi-directional differentiation potential,and can be converted into neurons when conditions permit.The lncRNA expression profile screened by high-throughput sequencing,IncRNA Database functional annotation combined with GO and KEGG enrichment analysis,initially predicted that IncRNA-Malatl is involved in regulating the transdifferentiation process of SGCs,and is related to the negative regulatory information of proBDNF.Therefore,this study explored the role and possible mechanism of IncRNA-Malatl and related transcription factors in regulating the transdifferentiation of satellite glial cells in the DRG.Method:After confirming the IncRNA related to this study through previous high-throughput sequencing analysis and Q-PCR verification,this study continued to cultivate DRG-SGCs,extract dorsal root ganglion tissue from newborn mice,and continue to culture to primary satellite gel Plasma cells migrated from the tissue,and then the cultured SGCs were divided into normal group and Anti-proBDNF intervention group(intervention dose 4ug/ml).At these 6 time points(24h,3d,7d,14d,21 d,28d)collect SGCs for the following experiments:Q-PCR,Elisa,cell immunofluorescence,overexpression and interference experiments in vitro.Results:1.The culture of satellite glial cells(primary)in the dorsal root ganglion is successful.2.Intervene with satellite glial cells and rat unilateral sciatic nerve injury model.through Anti-proBDNF serum.Q-PCR validates the pre-related target genes and predicts related transcription factors,which is consistent with the sequencing situation.3.Through the Elisa kit,it was discovered that SGCs can autocrine malatl,proBDNF/BDNF and their receptors,and their expression level changes during the process of transdifferentiation.The expression of malat1 in SGCs may affect the phenotypic change of glial cells and transdifferentiate in SGCs During the process,malat1 expression is affected by endogenous anti-proBDNF secretion.4.Construct the transfection experiment vector,and carry out preliminary.experiments for the overexpression vector and the interference vector respectively.The preliminary experiment shows that the lentiviral titer and dose provided in the kit can be used as the optimal value in the experiment.The results of the formal transfection experiment are consistent with the pre-experiment.5.Identification of immunofluorescence results.After overexpression and over-expression,the intervention of anti-proBDNF can reverse the effect of malat1 on cell apoptosis to a certain extent,and restore the number of cells to a certain extent;after interfering with malat1,The low expression of malat1 may cooperate with anti-proBDNF to regulate cell transdifferentiation.6.Q-PCR verification results showed that there were differential expressions of BDNF signaling pathway related factors and their receptors and transcription factors AKT1,NKX2.2,CTNNB1,FZD6 before and after the transfection experiment.Conclusion:1.After lncRNA expression profiling analysis and bioinformatics analysis,it was found that LncRNA-malat1 and its related transcription factors may be related to the process of SGC transdifferentiation and may play a role through the BDNF signaling pathway.2.There are malat1,BDNF/proBDNF and its receptor TrkB/p75NTR in normal dorsal root ganglion tissue and satellite glial cells,and their expression levels are affected by endogenous anti-proBDNF.3.1ncRNA-Malatl knockdown/overexpression may be regulated by proBDNF/P75NTR and BDNF/TrkB signals to affect the transdifferentiation of satellite glial cells in DRG.