The Study On MiR-22-3p Repairing Spinal Cord Sensory Conductive Function Via CBL/p-EGFR/p-STAT3/GAP43/p-GAP43 Pathway

Objective In this study,we investigated the effect of Epidermal Growth Factor(EGF)on primary sensory neuron neurite growth,and explored the effects of enhancing intrinsic growth capacity of adult primary sensory neurons on the recovery of sensory conductive function after spinal cord dorsal column injury(SDCI).miR-22-3p was regulated in primary sensory neurons in vitro and in vivo to observe the effects of miR-22-3p on axon growth and spinal cord sensory conductive function in primary sensory neurons,so as to find key targets and treatments to promote axon regeneration after SDCI.Method 1.Primary sensory neurons were extracted and cultured in vitro.Then the neurons were treated with drugs such as EGF,small interfering RNA(si RNA),miR-22-3p mimics,AG490,and Ruxolitinib to observe the differences in neuronal axon growth regulative effect and the expression level of signaling pathway proteins in cells.2.NF-200 Immunofluorescence staining was used to label and calculate the neurite length in order to compare the effects of EGF,si RNA,miR-22-3p mimics,AG490,and Ruxolitinib on the growth of primary sensory neuron neurites.3.The expression of phosphorylated Epidermal Growth Factor Receptor(p-EGFR),Casitas B-Lineage Lymphoma(CBL),Signal Transduction and Activator of Transcription 3(STAT3)phosphorylated STAT3(p-STAT3),Extracellular Regulated Protein Kinases1/2,Erk1/2),phosphorylated Erk1/2(p-ERK1/2),Growth Associated Protein 43(GAP43),and phosphorylated GAP43(p-GAP43)were determined by western blot assay in order to explore the intracellular signaling pathway involved in EGF promoting axon growth of primary sensory neurons.4.The miRDB,Star Base,Targetscan,and miRwalk databases were used to conduct bioinformatics predictions on miRNAs that potentially targeted CBL proteins.The interaction between miR-22-3p and the 3 ' UTR of CBL m RNA was verified using a dual-luciferase reporter assay.miR-22-3p and CBL m RNA were detected by real-time fluorescence quantitative polymerase chain reaction(RT-q PCR)to evaluate the inhibitory effect of miR-22-3p on CBL expression.5.Spinal cord dorsal column injury model was established and DRG injection was performed with miR-22-3p Agomir.The key proteins EGF,p-EGFR,CBL,p-STAT3 and p-GAP43 were detected in vivo using immunoblotting experiments to verify whether miR-22-3p can also regulate the CBL / p-EGFR / p-STAT3 / GAP43 / pGAP43 pathway in vivo.6.Somatosensory Evoked Potentials(SSEP)and Tape Removal Teat(TRT)were used to evaluate the integrity of the spinal cord ascending sensory pathway and the sensory function of rat hindlimbs after spinal cord dorsal column injury,respectively.Thus,the repair efficacy of miR-22-3p in DRG on sensory conducntive function after SDCI.Results 1.EGF promotes neurite growth in primary sensory neuronsEGF increased neurite growth in the 30 min and 1 h groups compared with that in the 0 mins group(P<0.05).However,a statistical difference was not observed between the 30 min and 1 h groups.Thus,prolonging the EGF treatment time exerted little further beneficial effect on neuronal axon outgrowth.The level of p-EGFR was increased in primary sensory neurons 30 mins after the EGF application.Interestingly,p-EGFR levels in 1h group was decreased compared with that in 30 min group.The decrease in p-EGFR levels in the 1 h group did not alter neurite growth compared with that in the 30 min group 2.CBL knockdown increases p-EGFR levels in cultured primary sensory neuronCBL expression was noticeably decreased in the CBL-si RNA group compared with that in the control group,which verified CBL si RNA knockdowned CBL efficiently.The p-EGFR expression was increased in CBL-si RNA group compared with that in control group.It is suggested that CBL is a key target to inhibit the expression of pEGFR.3.miR-22-3p is the potential miRNA that targets CBLThe potent miRNAs that targeted CBL were predicted by four databases,miR-22-3p was the common miRNAs predicted by the four databases.A dual-luciferase assay verified the interaction of miR-22-3p and the CBL m RNA 3'UTR.MiR-22-3p was expressed in primary sensory neurons and miR-22-3p mimics upregulated its expression.The expression of the CBL m RNA was noticeably inhibited in the miR-22-3p mimic-treated group.4.miR-22-3p increases neurite growth and p-EGFR levels in primary sensory neuronsThe level of the CBL protein was substantially decreased by the miR-22-3p mimic treatment.Meanwhile,p-EGFR levels were increased in the miR-22-3p mimics group.Thus,miR-22-3p removed the inhibitory role of CBL on p-EGFR.The miR-22-3p mimics significantly increased the neurite length compared with the control group 5.The p-STAT3/GAP43/p-GAP43 axis is downstream of p-EGFRThe pathway downstream of p-EGFR in EGF-treated primary sensory neurons remained unclear.STAT3 and Erk have been shown to be activated downstream of pEGFR.Levels of Erk1/2,p-Erk1/2,STAT3,p-STAT3,GAP43 and p-GAP43 were detected.Erk1/2 and p-Erk1/2 levels were not altered at all time points.The expressions of p-STAT3,GAP43 and p-GAP43 were increased at both 30 mins and 1 h.It was suggested that the p-STAT3/GAP43/ p-GAP43 pathway was the intracellular downstream pathway of p-EGFR.6.miR-22-3p /CBL/p-EGFR regulates neurite growth in primary sensory neurons via the p-STAT3/GAP43/p-GAP43 axisThe miR-22-3p mimics substantially promoted the elongation of neurites from primary sensory neurons compared with the control group,while AG490 and ruxolitinib blocked the effect of miR-22-3p.The levels of p-STAT3 and p-GAP43 were increased in the mimics group,and the AG490 and ruxolitinib treatments inhibited these changes.MiR-22-3p promoted neurite growth in primary sensory neurons through the CBL/p-EGFR/p-STAT3/GAP43/p-GAP43 axis.7.miR-22-3p regulates CBL/p-EGFR/p-STAT3/GAP43/p-GAP43 axis in vivoThe levels of EGF,p-EGFR and miR-22-3p were downregulated sharply at 7 d and increased gradually at 14 and 28 d after injury.The level of CBL was evidently inhibited and the level of p-EGFR,p-STAT3 and p-GAP43 were evidently promoted in miR-22-3p Agomir group.These results indicated miR-22-3p could also regulate CBL/p-EGFR/p-STAT3/GAP43/p-GAP43 axis in vivo.8.miR-22-3p promotes sensory conductive function recover after SDCLThe SSEP was detected to reflect the integrity of sensory conductive pathway.Tape Remove Test was conducted to show the sensory conductive function of hind paw.The SSEP and Tape Remove Test performance were restored after miR-22-3p Agomir treatment.hese results indicated miR-22-3p could restore hind paw sensory function via repair the injured sensory conductive pathway in spinal cord dorsal column.Conclusion 1.The limited promotion effect of EGF on primary sensory neuron neurite growth is due to the degeneration of p-EGFR operated by CBL.2.MiR-22-3p could target CBL to upregulate the level of p-EGFR and downstream p-STAT3/GAP43/p-GAP43 axis to promote primary sensory neuron neurite elongation.3.MiR-22-3p/CBL/p-EGFR/p-STAT3/GAP43/p-GAP43 axis would be a novel target to treat sensory disorders following spinal cord injury.