| Objective : Part one : By sequencing the genes of normal and PND mice hippocampus,we observed the changes of mi RNA expression profile in PND mice hippocampus,screened differentially expressed mi RNA,and predicted the corresponding target genes and their related functions through bioinformatics analysis.Part two:To evaluate the expression of mi R-320-3p in hippocampus of PND mice.Methods : Male C57/BL6 mice aged 12-14 weeks and weighing 20-25 g were selected.Part one: Six mice were randomly divided into two groups(n=3),namely control group(Con)and experimental group(Exp).The PND mouse model was established by internal fixation of tibia fracture under isoflurane anesthesia.Morris water maze test and open field test were conducted before and on the third day after operation.The latency of escape in water maze,the percentage of stay time in target quadrant and the total distance of open field movement were recorded respectively,and their spatial memory and autonomous activity ability were evaluated.At the end of the behavioral experiment three days after operation,we take out its hippocampus tissue after killing the mouse,and used for high-throughput gene sequencing.The differential mi RNA was analyzed by differential clustering analysis,mi Randa,target gene function analysis(GO),signal pathway enrichment analysis(KEGG)and other biological information.Part two:Ninety mice were randomly divided into three groups(n=30),namely normal control group(C),inhalation anesthesia group(A)and anesthesia operation group(S).The normal control group received no treatment,the inhalation anesthesia group continued isoflurane anesthesia for 15 min,and the anesthesia operation group received isoflurane anesthesia for intramedullary nail fixation of tibial fracture.Morris water maze test and open-field test were performed before operation and on the 1st,3rd and 7th day after operation.The latency of escaping from water maze,the percentage of staying time in target quadrant and the total distance of open-field movement were recorded respectively,and their spatial memory and autonomous activities were evaluated.On the 1st,3rd and 7th day after the operation,the hippocampus was sacrificed and the mRNA of mi R-320-3p and IGF-1 was detected by q RT-PCR,and IGF-1 was detected by western blot.Results : Part one:(1)Morris water maze and open field experiment showed that compared with Con group,the escape latency of Exp group was prolonged and the percentage of staying time in target quadrant was decreased 3 days after operation(P<0.05).There was no significant difference in the total distance of movement between the two groups(P>0.05).(2)Two groups of data were screened by high-throughput gene sequencing,Seven mi RNA were differentially expressed,among which mmu-mi R-320-3p,mmu-mi R-6236,mmu-mi R-6239,mmu-mi R-676-3p,mmu-mi R-1298-5p and mmu-mi R-344d-3p were all up-regulated,the expression of mmu-mi R-380-3p decreased;There are 783 overlapping target genes predicted by Target Scan and mi Randa database.In the functional enrichment analysis of GO target genes,the significant functions of differential genes are mainly enriched in transcriptional activator activity,protein serine/threonine activity,synaptic structure components,axon generation,mRNA catabolism and so on;Based on KEGG database,the significance analysis of Pathway involved by target genes showed that the differential genes were mainly concentrated in MAPK signaling pathway,RAS signaling pathway,m TOR signaling pathway,insulin signaling pathway and neurotrophic factor signaling pathway.Part two:(1)Morris water maze and open field experiment showed that compared with group C,the latency of escape in group S was prolonged and the percentage of stay time in target quadrant was decreased on the 1st,3rd and 7th day after operation(P < 0.05).Compared with group C there was no significant difference in group A(P > 0.05).There was no significant difference in the total movement distance among the three groups(P>0.05).(2)The mRNA of mi R-320-3p and IGF-1 were detected by QRT-PCR.Compared with group C,the mRNA expression of mir-320-3p in group S increased on the 1st and 3rd day after operation,while the mRNA expression of IGF-1 decreased on the 1st,3rd and 7th day after operation(P<0.05).Compared with group c,there was no significant difference in group a(P>0.05).(3)Compared with group C,the expression of IGF-1 protein in group S decreased on the 1st day after operation(P<0.05),but there was no significant difference on the 3rd and 7th day after operation(P>0.05).Compared with group c,there was no statistical difference in group a(P>0.05).Conclusion :(1)seven mi RNA including mi R-320-3p were significantly differentially expressed in the hippocampus of PND mice.(2)Through bioinformatics analysis,it is suggested that differential mi RNA may participate in the progression of PND disease through several signal pathways such as MAPK,RAS,m TOR,neurotrophic factor and insulin,which provides more research directions for exploring the pathogenesis of PND.(3)q RT-PCR verified that the changes of related RNA(mi RNA-320-3p,IGF-1mRNA)were consistent with the biological information analysis results,which proved the reliability of sequencing results and speculated that mi R-320-3p might participate in the occurrence and development of PND diseases by targeting IGF-1 mRNA. |
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