Effect And Mechanism Of MiR-335-5p On The Structure And Function Of Blood-spinal Cord Barrier

Background:Spinal cord injury(SCI)is a significant cause of death and long-term disability worldwide.The blood-spinal cord barrier is a physical barrier between the blood and spinal cord,which can prevent toxins,blood cells,and pathogens from entering the spinal cord.In the process of primary spinal cord injury,the blood-spinal cord barrier is disrupted,and various inflammatory factors,neutrophils,and macrophages infiltrate the spinal cord injury area through the barrier gap.Endothelial cells gradually lose their endothelial cell characteristics and transform into cells with mesenchymal cell characteristics under the infiltration of a large number of inflammatory factors.This process is called endothelial mesenchymal transition(EndoMT),This also leads to a further increase in the permeability of the blood-spinal cord barrier,with various inflammatory factors,neutrophils,and macrophages infiltrating the spinal cord injury area through the blood-spinal cord barrier,leading to secondary injury and neurological damage.MiRNA is a non-coding RNA molecule that negatively regulates the expression of genes,which can be used as a diagnostic biomarker and is becoming a new therapeutic target for central nervous system injury.In the early stage,through the analysis of the comprehensive data set of gene expression(GSE19890),it was found that miR-335-5P was significantly reduced after spinal cord injury.Then,through the mouse spinal cord injury model,it was confirmed that miR-335-5P was indeed significantly reduced after spinal cord injury,Previous studies have reported that miR-335-5p promotes the invasion and metastasis of colorectal cancer by promoting epithelial mesenchymal transition(EMT).As endothelial mesenchymal transition is a subcategory of epithelial mesenchymal transition,this study will investigate whether miR-335-5p affects the permeability of the blood-spinal cord barrier by regulating tight junction proteins(TJ proteins)and endothelial mesenchymal transition(EndoMT)during spinal cord injury,To provide new ideas and basis for using miR-335-5P as a new target to alleviate secondary injury and even treat spinal cord injury in the future.Objective:By establishing a three-dimensional external blood-spinal cord barrier(BSCB)model,we studied the effect of miR-335-5P on the permeability of the blood-spinal cord barrier(BSCB)by regulating tight junction protein(TJ protein),endothelial cell mesenchymal transformation(EndoMT)and endothelial cell activity.Methods:1.Transfect mimic NC,miR-335-5P mimic(enhancement group),inhibitor NC,miR-335-5P inhibitor(silence group)into immortalized mouse brain microvascular endothelial cells(Bend.3).2.Mouse brain microvascular endothelial cells(Bend.3)were seeded on the transwell cell to construct the blood-spinal cord barrier model in vitro.3.RT-qPCR was used to detect the expression of miR-335-5P and TGF of mouse brain microvascular endothelial cells(Bend.3)after spinal cord injury-β The expression of miR-335-5P arid transfection efficiency after treatment.4.Use CCK8 method to determine the TGF of mimic NC,miR-335-5P mimic,inhibitor NC,miR-335-5P inhibitor groups-β Cell viability under action.5.Western blotting and immunofluorescence were used to detect the expression differences of tight junction protein(ZO-1,Occludin)and mesenchymal cell markers(SNAIL,VIMENTIN,A-SMA)in each group.6.The permeability of blood-spinal barrier in vitro was measured by FITC-dextran flux in each group.Results:1.The relative expression of miR-335-5P in the miR-335-5P mimic group was significantly higher than that in the mimic NC group,and the miR-335-5P inhibitor group was significantly lower than that in the inhibitor NC group.2.The expression of miR-335-5P decreased significantly after spinal cord injury in mice,and the expression of TGF in mouse brain microvascular endothelial cells(Bend.3)-β The expression of miR-335-5P decreased significantly after treatment.3.MiR-335-5P mimic decreased the activity of mouse brain microvascular endothelial cells(Bend.3),while miR-335-5P inhibitor increased the activity of mouse brain microvascular endothelial cells(Bend.3).4.In the miR-335-5P mimic group,the expression of tight junction protein decreased and the expression of mesenchymal cell markers increased,while in the miR-335-5P inhibitor group,the opposite was true.5.Evaluate the in vitro blood-spinal cord barrier permeability of each group through the dye intensity in the upper to lower compartments of the transwell using FITC labeled dextran.Conclusion:miR-335-5P increases the permeability of the blood-spinal cord barrier by reducing the proliferation activity of brain microvascular endothelial cells,tight junction protein expression,and promoting endothelial mesenchymal transition(EndoMT),providing new targets and ideas for the treatment and treatment mechanism of secondary spinal cord injury.