MiR-137 Inhibited Inflammatory Response And Apoptosis After Spinal Cord Injury Via Targeting Of MK2

Objective: Spinal cord injury(SCI)is a common and troublesome disorder,results in placing a huge burden on human society and a tremendous impact to the mind and body of patients.Mitogen-activated protein kinase-activated protein kinase 2(MK2),which belongs to the MAPK family,is a direct substrate of p38 ? and p38 ? MAPKs.The expression and activated level of MK2 is related to the pathology and processes of thousands of inflammatory diseases..The predictive software predicted that there was a theoretical 7 base pairing binding region between miR-137 and MK2 3' UTR region.So does miR-137 play a protective role in spinal cord injury,and whether this effect can be achieved by inhibiting the expression level of MK2? There is no related report on the combination of miR-137 and MK2,the above scientific problems arouse our research interest.We define the differential expression of miR-137 and MK2 in serum of patients with SCI,and to explore its correlation;To identify the expression of miR-137 and MK2 in the glial cells(C8-DA1)and neurons(C8-B4),and its effects on inflammatory response and apoptosis.To clarify the negative regulation of miR-137 on MK2 and to clarify the targeting site;Through an animal model trial performed using mice,we try to demonstrate the protective effect of miR-137 on inflammatory response and apoptosis after spinal cord injury.The outcomes of the present study might indicate a new target in molecular treatment of SCI.Methods: 1.First collected serum from 19 patients with acute SCI and 19 patients without SCI.Realtime PCR was used to detect the expression of miR-137.Realtime PCR and Western blot was used to detect the expression of MK2.We also detected miR-137 and MK2 at the cellular level.The C8-DA1 and C8-B4 injury models were constructed by hydrogen peroxide induction method.The expression of MK2 in each cell model was detected.In the model,mimic control and miR-137 mimics were transfected in groups,and a control group was established.The expressions of inflammatory factors TNF-? and IL-6 in each group were detected by immunofluorescence staining,realtime PCR and western blot.The change of apoptosis in each group was detected by Tunel method.Differential expression of MK2 was detected by IHC,Western blot and realtime PCR and statistical analysis was performed.2.Overexpressing and silencing miR-137 were performed to detect the negative regulation of MK2.Construction of a wild-type and mutant miR-137 binding site MK2 reporter gene plasmid pmirGLO-MK2-wt and pmirGLO-MK2-mut,a dual luciferase reporter gene confirms the targeted binding of miR-137 to the MK2 3'UTR and clearly combine sites.Construct MK2 overexpressing vector plasmids pcDNA3.1-MK2-wt and pcDNA3.1-MK2-mut containing wild-type and mutant miR-137 binding sites.The constructed MK2 overexpressing vector plasmid and miR-137 mimics were co-transfected to detect the expression levels of MK2,TNF-? and IL-6.The apoptosis of the cells in each group after co-transfection was detected by Tunel method.3.The rat SCI animal model was constructed by Allen's method.Rat hind limb function score(Basso,Beattie,Bresnahan,BBB).The miR-137 intervention was performed 4 weeks(four groups: sham operation group,SCI control group,agomir control group,miR-137 agomir group).The BBB score was used to assess motor function after spinal cord injury in rats.The expressions of miR-137,MK2 and TNF-?/IL-6 in the spinal cord of each group were detected after 4 weeks.The apoptosis of spinal cord tissue of each group of animals after different interventions was detected by Tunel method.Results: 1.In serum specimens of patients,decreased miR-137 was presented in 84%(16/19)of serum specimens with SCI(P < 0.01),elevated MK2 was presented in 95%(18/19)of serum specimens with SCI.There was a negative correlation between the expression of miR-137 and MK2(P < 0.001).2.we also detected miR-137 and MK2 at the cellular level.A down-regulated miR-137 was found in hydrogen peroxide treated C8-D1 A and C8-B4 cells when compared with the control group,respectively(P < 0.01).MK2 at the cellular level was presented with an up-regulated tendency.we assessed the effect of miR-137 on inflammatory response after hydrogen peroxide intervention in C8-D1 A and C8-B4 cells.Compared with the control and mimic control group,transfection of miR-137 mimics led to a significant elevation of miR-137 in hydrogen peroxide treated C8-D1 A and C8-B4 cells.Up-regulation of miR-137 significantly reduced TNF-? and IL-6 expression in hydrogen peroxide intervened C8-D1 A and C8-B4 cells.2.Overexpression of miR-137 also inhibited apoptosis in hydrogen peroxide intervened C8-D1 A and C8-B4 cells.Through the online prediction software,we found that there was a theoretical 7 base-pair binding region for miR-137 in MK2 3'UTR.Up and down-regulation of miR-137 could negatively affect MK2 expression in both mRNA and the observed protein level.We constructed a luciferase reporter plasmid and the results of luciferase assay demonstrated a significant weakening of fluorescence in miR-137 mimics and pmirGLO-MK2-wt co-transfection group which indicated that MK2 was a target of miR-137.When we focused on the inflammatory response changes,we found that the inflammatory response was suppressed by elevation miR-137 while the suppression effect was reversed by pcDNA3.1-MK2-mut but not pcDNA3.1-MK2-wt.The inhibitive effect of miR-137 on apoptosis was also rescued by pcDNA3.1-MK2-mut but not pcDNA3.1-MK2-wt.3.Elevation of miR-137 by injection of a miR-137 agomir led to a faster and higher BBB score.The expression of MK2 was remarkably elevated in the control group and in the agomir NC injection group,while the facilitative effect was reversed by up-regulation of miR-137.Compared to the sham surgery group,the inflammatory response in the control group and in the agomir NC injection group were significantly activated.The apoptosis cells in the control group and in the agomir NC group were obviously more than that in the sham surgery group.Also,elevation of miR-137 presented a clear suppression effect on apoptosis.Conclusion: In the SCI patient serum and hydrogen peroxide-induced cell injury model,miR-137 was down-regulated and MK2 was highly expressed,and the expression of MK2 was negatively correlated.The increase of miR-137 expression could inhibit the inflammatory response and apoptosis;miR-137 can negatively regulate the expression and activity of MK2;miR-137 overexpression can attenuate the inflammatory response and apoptosis of rat spinal cord injury model;The results of this study may provide new gene-targeted therapy for SCI;...