| Objective: Neurons are permanent cells that are almost impossible to repair after injury.And axon regeneration is a sign of regeneration in injured neuron.Therefore,for the repair of neurons after injury,many studies are dedicated to how to promote axon regeneration.A large number of studies have shown that PI3K/PTEN/Akt/m TOR signaling pathway is an important regulator of axon regeneration.However,for damaged neurons,after PTEN knockdown or m TOR inhibition,which gene expression levels have been changed remains unclear.In summary,the study applied bioinformatics methods to identify the hub genes of damaged neurons after PTEN knockdown.Then we transferred si RNA to HT22 to interfere with PTEN expression or inhibited the activity of m TOR,explored which hub gene expression levels have been changed after PTEN inference or m TOR inhibition by molecular biological technology.Method: We searched the microarray datasets of the wild-type injury group and the postinjury PTEN knockdown group in the GEO database,and obtained DEGs through GEO2 R analysis.GO and KEGG analysis on the DAVID website was carried out to analyze gene enrichment and related signal pathways.Then STRING database was applied for PPI network analysis,and Cytoscape software was conducted to visualize and identify the common genes in three algorithms of MNC,MCC and DMNC as the hub genes.Finally,HT22 cells were transfected with si RNAs.Among control group,NC group and PTEN-si RNA group,we detected the interference efficiency of PTENsi RNA by RT-PCR technology,the protein expression levels of PTEN and p S6 k by Western Blotting and then PTEN and m RNA expression levels of hub genes by RTPCR/RT-q PCR technology.After that,we detected m RNA expression levels of hub genes by RT-PCR/RT-q PCR technology between control group and rapamycin group to prove the expression levels are opposite to the interference of PTEN.Result: After compared the wild-type injury group and the post-injury PTEN knockdown group,852 DEGs were obtained,of which 444 were up-regulated and 408 were downregulated.The GO items were mainly related to the structure and function of nervous system,including transmission of nerve impulse,synaptic transmission and phototransduction;ion channel activity and substrate specific channel activity in synapse and axon.The main pathways involved were neuroactive ligand-receptor interactions and substance metabolism.STRING database and Cytoscape software were used for PPI network analysis,and then Grk1,Shank1,Gria3,Cacng3,Gabrg1,Gad2,Cacna2d1,Nrn1,Cacng2 were identified as hub genes by selecting shared genes from three algorithms of MNC,MCC and DMNC.Compared with control group and NC group,RT-PCR result showed that the PTEN expression level of PTEN-si RNA1 group was significantly decreased(P<0.05).The Western Blotting result showed that PTEN was down-regulated and the expression of p S6 k increased to indicate m TOR signaling pathway was activated.The results of RT-PCR and RT-q PCR showed that the expression levels of Grk1,Cacna2d1 and Nrn1 were consistent with the predicted,but Gria3 was not.Then,the results of RT-PCR and RT-q PCR showed that the expression levels of Cacna2d1 and Nrn1 in the rapamycin group decreased compared to control group,which was reversely confirmation of the predicted change levels.Conclusion:(1)Bioinformatics analysis identified that hub genes after PTEN knockdown in injured neurons were Grk1,Shank1,Gria3,Cacng3,Gabrg1,Gad2,Cacna2d1,Nrn1,Cacng2.(2)The expression levels of Cacna2d1 and Nrn1 were consistent with the predicted levels after PTEN knockdown or m TOR inhibition,which could indicate that Cacna2d1 and Nrn1 were downstream key genes to promote axon regeneration of injured neuron after PTEN knockdown. |
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