Mechanism Of LncRNA MALAT1 Targeting MiR-124-3p Regulating Calpain 1 Inhibiting Neuronal Apoptosis After Spinal Cord Injury

ObjectiveSpinal cord injury(SCI)often causes permanent partial or even complete impairment of sensory and motor functions in patients.Currently,there is no effective way to treat SCI,and further exploration of the occurrence,progression,and outcome of SCI is needed to provide new targets and directions for the diagnosis and treatment of SCI.Competitive endogenous RNA(ce RNA)plays an important role in central nervous system diseases,but its research in SCI is unclear.In this experiment,we investigated the effects of the ce RNA interaction network composed of lnc RNA MALAT1,miR-124-3p,and calpain 1 on SCI,and explored the interaction between them,hoping to provide new targets and directions for the treatment of SCI.Method1.After establishing a rat SCI animal model,the BBB behavioral score was used to evaluate the success of the modeling.RT-q PCR was used to detect the changes in lnc RNA MALAT1,miR-124-3p,and calpain 1 in each group of rats.2.The relationship between miR-124-3p and lnc RNA MALAT1 was predicted by "starbase",and the targeting relationship between miR-124-3p and lnc RNA MALAT1 was verified by double luciferase experiment.3.Different concentrations of H2O2 acted on PC12 cells for 24 hours,and the best conditions for SCI modeling were found through CCK8 cell viability test.The optimal concentration of H2O2 was applied to PC12 cells for 24 hours,and the expression of miR-124-3p,lnc RNA MALAT1 and calpain 1 were measured by RT-q PCR.Then the changes of CAPN1 and cleared caspase 3 were measured by WB.4.PC12 cells were directly transfected with miR-124-3p,and the expression of miR-124-3p was determined by RT-q PCR.The cells were divided into control group,H2O2 group,miR-124-3p+H2O2 group and miR-NC+H2O2 group.The cell viability,apoptosis rate and changes of CAPN1 and cleared caspase 3 were measured and calculated by CCK8,flow cytometry and WB.5.Three different interference fragments of lnc RNA MALAT1 were constructed.RT-q PCR was used to detect the interference efficiency and screen the most suitable interference fragments.They were divided into control group,H2O2 group,siMALAT1+H2O2 group and si-NC+H2O2 group.The effects of interference with lnc RNA MALAT1 on cell viability and apoptosis rate were analyzed by CCK8 and flow cytometry.6.The lnc RNA MALAT1 and miR-124-3p were simultaneously interfered,and the experiment was divided into control group,H2O2 group,si-NC+H2O2 group,siMALAT1+H2O2 group,si-MALAT1+inhibitor NC+H2O2 group,and siMALAT1+miR-inhibitor+H2O2 group.The cell viability,apoptosis rate,and changes in CAPN1 and cleared caspase 3 were measured and calculated by CCK8,flow cytometry and WB.Results1.lnc RNA MALAT1,miR-124-3p,and calpain 1 are differentially expressed in SCI rat animal models.2.The prediction result of "starbase" shows that miR-124-3p is the target of lnc RNA MALAT1.The double luciferase experiment verifies the correlation between lnc RNA MALAT1 and miR-124-3p.3.CCK8 experiment screened the best conditions for modeling,and the results showed that when the concentration of H2O2 was 200 μM,PC12 cells were stimulated for 24 hours to obtain the median lethal dose,which was used as the condition for simulating the apoptosis model of SCI in vitro.WB and RT-q PCR results showed that the expression of miR-124-3p in SCI cell model decreased significantly,while the expression of calpain 1,lnc RNA MALAT1 and cleared caspase 3 increased significantly.4.After miR-124-3p transfection,the RT-q PCR results showed that the expression of miR-124-3p was significantly up-regulated.The results of CCK8 and flow cytometry showed that miR-124-3p could promote cell viability and inhibit cell apoptosis after SCI.WB results suggested that miR-124-3p significantly inhibited the expression of CAPN1 and cleared caspase 3.5.Three interference fragments of lnc RNA MALAT1(si-1,si-2 and si-3)were constructed,and the best fragment si-1 was screened by RT-q PCR.Through CCK8 and flow cytometry,it was found that knockdown of lnc RNA MALAT1 could inhibit cell apoptosis induced by SCI and promote cell proliferation.6.200 μM H2O2 induced co-transfection of si-lnc RNA MALAT1 and miR-124-3p inhibitor cells for 24 h.RT-q PCR results suggested that interfering with lnc RNA MALAT1 could promote the expression of miR-124-3p,while miR-124-3p inhibitor could block this effect.Then,CCK8 and flow cytometry detection showed that miR-124-3p inhibitor reduced the number of cells rescued by interfering with lnc RNA MALAT1,thus inhibiting cell viability.WB test also found that miR-124-3p inhibitor inhibited the effect of si-lnc RNA MALAT1 on CAPN1 and cleared caspase 3 protein levels.Conclusion1.Overexpression of miR-124-3p inhibits neuronal apoptosis after SCI by targeting calpian 1;2.Knockdown of lnc RNA MALAT1 can inhibit neuronal apoptosis after SCI;3.lnc RNA MALAT1 can inhibit SCI-induced neuronal apoptosis by targeting miR-124-3p to regulate calpain 1.