| ObjectiveNeuropathic pain(NP)is poorly managed,and in-depth mechanisms of gene transcriptome alterations in NP pathogenesis are not yet fully understood.As a non-drug intervention,exercise training is an important research direction in the treatment of neuropathic pain,but its mechanism remains unclear.micro RNAs(miRNAs),owing to their high stability and evolutionary conservation,are expected to be used as potential therapeutic targets or diagnostic biomarkers.Hence,this study was aimed to 1.determine micro RNA-related molecular mechanisms of NP and their transcriptional;2.determine transcriptome changes in dorsal root ganglia(DRG)of mice with neuropathic pain;3.clarify the effects of aquatic exercise training on the expression of miR-183 cluster and its target genes in DRG and allodynia alleviation of SNI mice;4.verify whether aquatic exercise training induced reversed expression of miR-183 and its target gene Cacna2d1/2 in peripheral neuropathic pain mice.MethodsThis study consists of four parts.In the first part of the study,Pub Med,Embase,Web of Science and CINAHL Complete(EBSCO)were searched from inception to April 2021.Commonly dysregulated miRNAs in NP were assessed.The putative targets of these miRNAs were determined using Target Scan,Funrich,Cytoscape and String database.In the second part,C57BL/6 mice were used for peripheral neuropathic pain model of spared nerve injury(SNI),and were randomly divided into SNI group(n=6)and sham-operated group(n=6).Mechanical withdrawal threshold(MWT)was conducted before surgery and on the 3rd,7th,14th,21st,28th,35th and 42th postoperative day.After 6 weeks,L4-L6 DRGs tissue was taken for RNA-sequencing,and the differentially expressed genes(DEGs)were determined through bioinformatics analysis.Then,the GO and KEGG enrichment analysis were performed on DEGs.Finally,combined with the results of part 1,the key DEGs were identified.In the third part,C57BL/6 mice were randomly divided into Na(?)ve group(n=15),sham group(n=15),sham+swim group(n=15),SNI group(n=15)and SNI+swim group(n=15).The sham+swim group and SNI+swim group received aquatic exercise intervention for 6 weeks,and the other groups received regular feeding.MWT tests were performed pre-and post-operatively.Morphological changes of ipsilateral DRGs and sciatic nerve were determined by HE staining.The expression changes and position of Cacna2d1 and Cacna2d2 were evaluated by immunofluorescence double staining.q RT-PCR and western blot(WB)were used to determine the m RNA and protein expression levels of miR-183 cluster(miR-183/182/96)and Cacna2d1/2 in L4-L6 DRGs.In the fourth part,the binding sequence of miR-183 and Cacna2d1/2 was predicted.Then double luciferase reporter gene assay experiment was conducted to verify the targeting relationship between miR-183 and Cacna2d1/2.Then,miR-183 knockout(KO)mouse model was constructed.They were divided into the miR-183 KO group,miR-183 KO+swim group,na(?)ve group and na(?)ve+swim group.The miR-183 KO+swim group and na(?)ve+swim group received aquatic exercise intervention for 6 weeks,and the other groups received regular feeding.MWT tests were performed pre-and post-operatively.The m RNA and protein expression levels of miR-183 cluster(miR-183/182/96)and cacan2D1/2 were detected by q RT-PCR and WB.ResultsIn the first part of the study,a total of 133 literatures containing miRNA profiles studies and experimentally verify studies were included.Venn analysis,target gene prediction analysis and functional enrichment analysis indicated several miRNAs(miR-200b-3p,miR-96,miR-182,miR-183,miR-30b,miR-155 and miR-145)and their target genes involved in known relevant pathways for NP.Targets on transient receptor potential channels,voltage-gated sodium channels and voltage-gated calcium channels may be harnessed for pain relief.In the second part,SNI model was successfully established.From 3 days postoperatively,the MWT of SNI mice was significantly lower than that of the Sham group(p<0.001),and remained at a low level for the following 6 weeks.Transcriptome sequencing showed that there were 1520DEGs(822 significantly up-regulated and 698 significantly down-regulated)in DRG of SNI mice.The intersection of these 1520 DEGs with the target genes of miRNAs verified in NP surgery model revealed that there were 8 overlapping genes.They are Cacna2d1,Csf1,Socs1,Bdnf,Irak1,Cacna2d2,Kcna2 and Akt3.GO analysis and KEGG analysis revealed that DEGs genes were significantly enriched in the classifications of axon development,axonogenesis,extracellular matrix,axon part,ion gated channel activity,glycosaminoglycan binding and Neuroactive ligand-receptor interaction pathway(p<0.05).In the third part,the MWT of SNI+Swim group was always significantly lower than that of shan+swim group(3d,7d,14d,21d,35d:p<0.001;42d:p=0.031),but after aquatic exercise intervention,MWT of SNI+swim group increased gradually,and it was significantly higher than that of SNI group at 21d(p=0.046),28d(p<0.001),35d(p<0.001)and 42d(p<0.001)after surgery.HE staining showed that the nerve fiber space of the sciatic nerve in the SNI+swim group was improved;the axon was clear and the proliferation of Schwann cells was reduced.In the SNI group,the DRG neurons showed vacuolar degeneration,the number of nisseni in the cells decreased,the morphology was unclear,and the morphology of nerve fibers was incomplete,which was significantly improved after aquatic exercise.Cacna2d1/2 fluorescence was significantly enhanced in DRG neurons in SNI group and normalized after aquatic exercise.q RT-PCR and WB results showed that the expressions of miR-183,miR-182 and miR-96 in SNI group were significantly decreased(p<0.05),while the m RNA and protein expressions of Canca2d1 and Cacna2d2 were significantly increased(p<0.001).After 6 weeks of aquatic exercise intervention,the expression of miR-183/182/96 in SNI+swim group was significantly increased(p<0.001)and the expression of Canca2d1/2 was significantly decreased(p<0.05)compared with that in SNI group.In the fourth part,the results of double luciferase reporter gene assay showed that mmu-miR-183-5p did not significantly inhibit the fluorescence expression of psi CHECK-Cacna2d1 and psi CHECK-Cacna2d1-mut(p>0.05),but significantly inhibited the fluorescence expression of psi CHECK-Cacna2d2(p<0.001).There was no significant inhibition on the fluorescence expression of psi CHECK-Cacna2d2-MUT(p>0.05).After 6 weeks aquatic exercise intervention,compared with the naive group,the MWT of the miR-183 KO+swim group was always significantly lower than that of the na(?)ve group(p<0.001).However,compared with miR-183 KO group,the MWT of miR-183 KO+swim group increased gradually,and from day 21,the MWT of miR-183 KO+swim group was significantly higher than that of miR-183 KO group(p<0.05).q RT-PCR results showed that the m RNA expression levels of Cacna2d1 and Cacna2d2in miR-183 KO group were significantly increased compared with naive group(p<0.05).After 6weeks of aquatic exercise,the m RNA expression levels of Cacna2d1 and Cacna2d2 in miR-183KO+swim group were significantly decreased(p<0.05).WB results showed that the expression levels of CACNA2D1 and CACNA2D1 in miR-183 KO group were significantly increased(p<0.05).After exercise,the expression level of CACNA2D1 in miR-183 KO+Swim group was significantly lower than that in miR-183 KO group,while there was no significant difference in CACNA2D2(p>0.05).Conclusion1.several miRNAs(miR-200b-3p,miR-96,miR-182,miR-183,miR-30b,miR-155 and miR-145)and their target genes involved in known relevant pathways for NP.Targets on transient receptor potential channels,voltage-gated sodium channels and voltage-gated calcium channels may be harnessed for pain relief.2.822 m RNAs were significantly up-regulated and 698 down-regulated in DRG of SNI mice.Combined with part 1,several overlapping differentially expressed genes were compared and analyzed,which were Cacna2d1,Csf1,Socs1,Bdnf,Irak1,Cacna2d2,Kcna2 and Akt3.Cacna2d1and Cacna2d2 belong to Ca _Vsα2δsubunit.These differential genes may be potential targets for the treatment of neuropathic pain3.Aquatic exercise can normalize mechanical stimulation hyperalgesia in peripheral neuropathic pain mice,and reverse the inhibition of miR-183 cluster expression and the up-regulation of Cacna2d1/2 expression caused by nerve injury.4.Cacna2d2 is the target gene of miR-183.miR-183 KO can induce mechanically-stimulated hyperalgesia in mice and up-regulate the expression of Cacna2d1 and Cacna2d2.Aquatic exercise could alleviate hyperalgesia in miR-183 KO mice to a certain extent and reduce the expression of Cacna2d1 but not Canca2d2.These results suggest that aquatic exercise training induced reversed expression of miR-183 and its target gene Cacna2d1/2 in peripheral neuropathic pain mice. |
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