Microrna-21-5p Targets ACVR2B In Mouse Neurons And Affects Axonal Regeneration After Spinal Cord Injury

Background:Traumatic Spinal cord injury(SCI)often causes permanent physical disability and has devastating consequences for the society,families and individuals.Two main reasons lead to the failure of nerve function recovery after traumatic injury of central nervous system(CNS):One is the regeneration inhibitory microenvironment after injury,the other one is insufficient regeneration capacity of neurons?The internal mechanism of neuron regeneration failure has not been clearly studied,so there is no effective therapies to completely cure spinal cord injury.microRNAs is a kind of endogenous non-coding RNA,it regulate a wide range of physiological processes.miR-21-5p(miR-21)has the nerve protective effect,a growing number of studies found that microRNAs play an important role in axon growth,bifurcation,and regeneration,but the function and its mechanism of miR-21 to axon regeneration after spinal cord has not well studies.Objective:To explore changes of microrma-21a-5p after traumatic spinal cord injury and the role of mir-21a-5p in axonal regeneration of spinal cord neurons through the in vitro injury model of primary neurons of mice,and the mechanism of mir-21a-5p in the pathological process of spinal cord injury was explored to provide theoretical basis and clinical basis for the treatment of spinal cord injury.Methods:The primary hippocampal neurons of mice were extracted,inoculated on a cover glass coated with Poly-L-lysine,and cultured in a 24-well plate.The neuron injury model was established in vitro to detect the mRNA expression changes of mir-21a-5p.After primary neurons were injured,they were divided into three groups:group one was cultured with normal medium,groups two were cultured with medium supplemented with Activin A,and groups three were cultured with Activin A after mir-21 mimic were used.The axon regeneration was examined by immunofluorescence staining two days after injury.The expression level of Acvr2b protein was detected after injury of neuron cell Ht22.Through up-regulation and down-regulation of mir-21,the expression of axonal regeneration related gene:gap-43,Sprrla,Galanin and the expression changes of gap-43 protein were detected,so as to clarify the effect of mir-21 on axonal regeneration.The cells were treated with pathway agonists and inhibitors to detect whether mir-21 regulates the Activin pathway and explore the mechanism of mir-21 on the pathway.Result:The purity of neuron cells was over 90%.The expression of mir-21 increased after the neurons injury,and cause decrease of Acvr2b mRNA and protein expression.Inhibition of mir-21 expression can up-regulate the expression of axonal regeneration related genes and significantly increase the axonal regeneration of neurons after injury compared with the control group.Activin A can significantly enhance the phosphorylation of downstream proteins in the Activin pathway and the up-regulation of mir-21 can inhibit the phosphorylation of downstream proteins in the Activin pathway.The results of immunofluorescence staining showed that the increase of mir-21 can inhibit the promoting effect of Activin A on axon regeneration after injury.After inhibiting mir-21,Activin A can significantly increase the phosphorylation of pathway proteins,which can be blocked by pathway inhibitors.Activin A can promote the expression of Gap-43,and pathway inhibitor can inhibit the expression of Gap-43.Conclusion:miR-21 expression was significantly increased after neuronal cell injury and attenuate the phosphorylation of downstream protein of the Activin pathway,smad2/3,by inhibiting Acvr2b.miR-21 also inhibited the regeneration of neuronal axons after injury,which may provide a potential therapeutic target for spinal cord injury.