A Study On The Mechanism Of MiR-99b-5p Regulation Of Akt/mTOR/STAT3 Signaling Pathway For Repairing Spinal Cord Injury

?Objective?Spinal cord injury?SCI?can cause abnormal changes in patients'sensation and motor function,which is one of the most serious diseases in spine surgery,and the treatment for SCI is also a medical problem.There are a lot of experimental studies suggest miR-99-b-5p may function in mammals rapamycin target protein?mammalian target of rapamycin,mTOR?,confirmed mTOR signaling pathway is closely related to the nerve repair of spinal cord injury,but the specific mechanism is unclear.So this research want to explore the mechanism of the miR-99-b-5p regulating Akt/mTOR/STAT3 signaling pathway to repair spinal cord injury by establishing the cell and animal model of spinal cord injury,and provide new theory and direction for the clinical treatment of spinal cord injury.?Method?In vitro experiment:the preparation of nerve cells of spinal cord injury,this experiment adopts the latest NYU IMPACTOR MODEL-?spinal cord strike device.After 1 week postoperative,take injury spinal cord segmental,digestion,separation,extraction and culture motor neuron cells.This experiment was divided into 3 groups,injury motor neurons as injury group?SCI?,injury nerve cells and miR-99-b-5p inhibitors as inhibitor group?SCI-miR?,culture spinal motor neurons of adult mices as the control group?Control?.Through the analysis of the RT-QPCR,cell transfection,cell apoptosis,dual luciferase report analysis and immunofluorescence and western blot analysis to observe miR-99-b-5p inhibitors effects on injury spinal cord nerve cells.In vivo experiment:30 female ICR mice?6 weeks?,weighing about 30 g,were randomly divided into three groups,with NYU IMPACTOR MODEL-?spinal cord strike device preparing spinal cord injury model.three groups:control group?only do lamina resection,no damage to the spinal cord,injection RNase-free H₂O??Control?,spinal cord injury group?spinal cord stroke injury,injection RNase-free H₂O??SCI?,inhibitor group?spinal cord stroke injury,injection miR-99-b-5p inhibitors??SCI-miR?.Observing behavior score of three groups of mice respectively after operation to observe the mice hind limbs motor function recovery,this experiment adopts the specialized score for mice BMS?Basso Mouse Scale?score.Postoperative2 week,3 groups spinal specimens were taken for immunohistochemistry and western blot analysis,detection of spinal cord cells markers glial fibrillar acidic protein?glial fibrillary acidic protein,GFAP?and Neurofilament protein 200?Neurofilament NF200?,nest protein?Nestin?and neuron nucleoprotein?Neuronal nuclei,NeuN?,etc.,and pathways of Akt/mTOR/STAT3 protein expression.?Result?In vitro experiments:The mRNA level of miR-99b-5p?NO.10 days?in injury nerve cells was higher than in the 0 day after spinal cord injury?P<0.05?,while the control group showed little difference.In the cells transfected with mTOR-WT plasmid,luciferase activity in the control group was higher than that in the inhibitor group?P<0.05?.In the experiment of apoptosis,the apoptosis of the inhibitor group was decreased,and the three groups were statistically significant?P<0.05?.In immunofluorescence experiment,the nerve axonal length of inhibitor group was increased?P<0.05?.miR-99b-5p inhibitor increases the mTOR protein level of spinal cord injury,and the mRNA content expression of mTOR is also increased?P<0.05?.In vivo experiment:The BMS score of the injury group and the inhibitor group increased over time,but the BMS score of the inhibitor group was better than that of the injury group?P<0.05?.Immunohistochemical explain that the inhibitor group center GFAP positive staining astrocytes of spinal cord injury number below the injury group,and presencing statistical differences?inhibitor group of GFAP positive cells was 14.19±2.51%,P<0.05?;The number of NF200 staining positive cells in the spinal cord injury center of the inhibitor group was increased?P>0.05?.Nestin staining showed that the number of positive cells in the inhibitor group increased?IOD:24808±4448.33,P<0.05?.Neun staining showed that the number of positive cells in the inhibitor group was more than that of the injured group?26.62±6.73%,P<0.05?.miR-99b-5p inhibitor up-regulated the protein expression in the Akt/mTOR/STAT3 signaling pathway,and there was statistical significance between the three groups?P<0.05??Conclusion?This study show that miR-99-b-5p act on mTOR signaling pathways,repairing injury spinal cord neurons by inhibiting nerve cell apoptosis,inhibiting the growth of astrocytes and promoting the growth of axons.miR-99-b-5p inhibitors can up regulate Akt/mTOR/STAT3 signaling pathway,benefite to the recovery of hind limbs function in SCI mice,inhibit the apoptosis of damage neurons in the spinal cord tissue,inhibit the glial scar formation,promote the growth of axons.So the application of miR-99-b-5p inhibitors up regulate Akt/mTOR/STAT3 signaling pathway and it may be a new approach for the treatment of spinal cord injury.