Expression And Function Of MiR-1246 In Secondary Spinal Cord Injury

BackgroundSpinal Cord Injury（SCI）is an extremely serious injury to the central nervous system in the current society,and is considered an incurable injury until today.People with spinal cord injury suffer greatly both physiologically and psychologically,and at the same time,with increasing prevalence,spinal cord injury has been a major burden on society.Various studies and clinical trials have been conducted,some of which have shown good results,but the long-term efficacy of SCI patients remains unsatisfactory and there is still no gold standard treatment with sufficient evidence.MicroRNAs are a class of endogenous non-coding single-stranded RNA molecules that participate in important life processes such as reproductive development,tumor development,and SCI repair by acting on target genes.As a member of Micro RNAs,miR-1246 is widely involved in many related studies,including inflammation,tumorigenesis and development,cell aging,and promotion of astrocytes.plasmoma cell proliferation and invasion,acute lung injury and other related diseases.Studies have shown that miR-1246 has been found in spinal cord injury rat tissues,but its expression and role in spinal cord injury are still unclear.ObjectiveThis study will explore the expression level of miR-1246 in spinal cord injury from the clinical,animal and cytological levels,and clarify the role of miR-1246 in secondary spinal cord injury.Method1.Collect 40 SCI patients’serum samples and 40 non-SCI patients’serum samples,RT-qPCR（Quantitative Real-time PCR,real-time fluorescence quantitative PCR）method to detect miR-1246 in the serum of the above two groups of patients.expression;2.Allen blow method was used to construct a rat model of spinal cord injury,and SD rats were randomly divided into control group（control group,without any operation）,sham group（mock surgical group,only the lamina and spinous process were removed）and SCI group（Spinal cord injury group）.BBB score and ramp test were used to evaluate the changes of motor and neurological function of the rats in each experimental group;immunohistochemical staining was used to investigate the expression of inflammatory marker TNF-αin the spinal cord injury site of the rats in each experimental group,and HE staining was used to evaluate the spinal cord injury of the rats in each experimental group The morphological changes of the parts were detected,and the differential expression of miR-1246 in the constructed rat SCI model and the other two groups was detected by RT-qPCR;the expression of miR-1246 was reduced by silencing the virus,and SD rats were randomly divided into sham group and SCI group.The BBB scoring system and other related functional tests were used to study the function and mechanism of miR-1246 in the treatment and repair of SCI.3.The PC-12 cells were cultured,and the cell model of spinal cord injury was constructed by H₂O₂stimulation method（H₂O₂group）,and the control group（NC group）was set simultaneously;RT-qPCR method was used to detect the inflammatory markers tumor necrosis factorα（TNF-α）and The expression of interleukin-6（IL-6）,and the differential expression of miR-1246 in the spinal cord injury cell model was detected by RT-qPCR.4.Transfect miR-1246 inhibitor（miR-1246 inhibitor）in the H₂O₂stimulation method to construct the spinal cord injury cell model to establish the H₂O₂+miR-1246inh group,and set the transfection control simultaneously（H₂O₂+NCinh group）;RT-The expression differences of inflammatory markers TNF-αand IL-6 in the cells of each group were detected by qPCR.Results1.The expression of miR-1246 in SCI patients and non-SCI patients serum samples:Compared with the non-SCI group,the relative expression of miR-1246 in the SCI group was significantly increased,and the difference was statistically significant（P<0.01）.2.The expression of miR-1246 in animal spinal cord tissue:（1）Compared with the control group and the Sham group,the behavioral and morphological results of the SCI group showed obvious spinal cord injury,indicating that the SCI model was successfully constructed.（2）The expression of miR-1246 in spinal cord tissue of SCI group was significantly higher than that of control group and Sham group,and the differences were statistically significant（P<0.01）.（3）The results of the SCI+miR-1246 interference group in terms of neurological function and motor function of spinal cord injury rats showed that the neuromotor recovery effect of the interference treatment group was significantly better than that of the SCI group（P<0.05）,indicating that the expression of miR-1246 was reduced.It has good curative effect on the treatment and repair of SCI.3.The expression of miR-1246 in H₂O₂-stimulated PC-12 cells:（1）Compared with the NC group,the relative expression of miR-1246 in the H₂O₂group was significantly increased,and the expressions of inflammatory markers TNF-αand IL-6 were also significantly increased,and the difference was statistically significant（P<0.01）;（2）Expression of TNF-αand IL-6 in each group after transfection with miR-1246 inhibitor:（1）Compared with the NC group,the expressions of TNF-αand IL-6 in the H₂O₂group were significantly increased.The expressions of-αand IL-6 increased,but the increase was still lower than that of the H₂O₂group.Compared with the above groups,the difference was statistically significant（P<0.05）.（2）Compared with the H₂O₂group,the relative expressions of TNF-αand IL-6 in the H₂O₂+NCinh group had no significant difference;the expression levels of TNF-αand IL-6 in the H₂O₂+iR-1246 inh group were significantly decreased,and the difference was statistically significant Academic significance（P<0.05）.Conclusion1.The expression of miR-1246 was increased in the serum of patients with spinal cord injury.2.The expression of miR-1246 was increased in the spinal cord tissue of the constructed SCI rat model;down-regulation of the expression of miR-1246 could promote the recovery of motor function.3.The expression of miR-1246 was increased in H₂O₂-stimulated PC-12 cells;down-regulation of miR-1246 in H₂O₂-stimulated PC-12 cells could reduce the expression of TNF-αand IL-6.