| Spinal cord injury is a serious nervous system disease with high morbidity and disability rate that can cause motor and sensory dysfunction in patients.In addition to motor dysfunction,patients will experience symptoms of ascending sensory beam damage such as chronic neuralgia,sensory disturbance,sensory dullness,and sensory loss after SCI,leading to cognitive and psychological disorders.At present,there is no effective treatment method for sensory conduction dysfunction after spinal cord injury,and there are few studies focus on the repair of injured spinal cord ascending sensory pathway.Effective treatment strategies to restore the sensory function and improve the life quality of SCI patients are urgently needed.Recent years,the discovery of micro RNA(miRNA)has provided the possibility for precise regulation of gene expression and signal transduction pathway targets.miRNA can adjust the expression of many key genes through post-transcriptional regulation mechanisms to change the prognosis of many diseases.miRNA usually interact with its target genes and target proteins to form a network regulatory mechanism.Previous research of our group have demonstrated that miRNAs can regulate the phenotype of primary sensory neurons(PSNs)through various forms,promote the repair of spinal ascending sensory fibers,and participate in complex molecular biological process such as neural cell differentiation,maturation,and neuromodulators release.Objective:The purpose of this study is to demonstrate miR-30 b could regulate the primary sensory neuron axon growth and recovery dorsal column sensory conduction function of injured spinal cord via the Sema3A/NRP-1/Plexin A1/ RhoA/ROCK pathway through in vitro and in vivo experiments.Methods:1.Four adult female Wistar rats(250±10g)were used to obtain primary sensory neurons for in vitro experiments.36 adult female Wistar rats(250±10g)were randomly divided into 3 groups(sham group(T10 laminectomy only),Scramble group(spinal cord dorsal column lesion+scramble)and Agomir group(spinal cord dorsal column lesion+miR-30 b agomir))for in vivo experiments.2.Bioinformatics was used to predict miRNAs which targeting sema3 A through MIRNAMAP,TARGETSCAN,STARBASE,and MIRWALK2 database.3.Dual-luciferase reporter assay was used to verify that sema3 A is a target gene of miR-30 b.RT-q PCR was used to detect sema3 A m RNA to demonstrate whether miR-30 b regulates sema3 A through inhibition or degradation.4.miR-30 b agomir or antagomir transfection was performed in primary sensory neurons.Co-immunoprecipitation experiments confirmed that Sema3 A binds to the NRP-1/Plexin A1 co-receptor.Western blot was used to detect Sema3 A,Sema3A/NRP-1 complex,Sema3A/Plexin A1 complex,GTP-RhoA,and ROCK protein in each group.Image-pro plus 6.0 was used to detect the axon length of each group to determine the effect of miR-30 b on the key pathway proteins and the axon length.5.Apply small interfering RNA(si RNA)to suppress the expression of sema3 A,while suppress miR-30 b,to demonstrate that sema3 A is a key target gene for miR-30 b regulating axon growth.6.After applying miR-30 b antagomir or adding Sema3 A to PSNs,RhoA/ROCK pathway inhibitor Y-27632 was added,and then key proteins and axon length of the pathway were tested to prove that miR-30 b and sema3 A regulate the neurite growth via RhoA/ROCK pathway.7.We performed immunofluorescence on primary sensory neurons and spinal cords to observe and compare the status of neurons and spinal cords in different groups.8.Nogo A-Fc was used to simulate the axon growth inhibitory microenvironment.We divided the PSN into Nogo A-Fc group and miR-30 b agomir & Nogo A-Fc group,and tested the axon length of each group to verify that miR-30 b can promote the growth of PSN axons in an inhibitory microenvironment.9.The somatosensory evoked potentials and tape remove test were used to verify the sensory function recovery of spinal cord dorsal column lesion rats in vivo.Results:1.miR-30 b promotes axonal growth of primary sensory neurons.2.miR-30 b inhibit sema3 A expression by inducing degradation of sema3 A m RNA in primary sensory neurons.3.miR-30 b regulates Sema3A/NRP-1/Plexin A1 complex formation by targeting sema3 A in primary sensory neurons.4.sema3 A is the key target gene for miR-30 b regulating axon growth of primary sensory neurons.5.RhoA/ROCK is the key downstream pathway of miR-30b/Sema3A/ NRP-1/Plexin A1 axis to regulate axon growth in primary sensory neurons.6.miR-30 b promotes primary sensory neurons axon growth in a Nogo AFc inhibitory microenvironment.7.miR-30 b could regulate the Sema3A/NRP-1/Plexin A1/RhoA/ROCK pathway of spinal cord dorsal column lesion rats.8.miR-30 b promotes sensory conduction recovery of rats with injured spinal cord dorsal column.Conclusion:miR-30 b can inhibit sema3 A autocrine by degrading sema3 A m RNA,and then promote the growth of primary sensory neuron axons in the inhibitory microenvironment through the NRP-1/Plexin A1 co-receptor and intracellular RhoA/ROCK pathway and then repairing sensory conduction function of spinal cord dorsal column lesion rats. |
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