MiR-872-5p/FOXO3a/Wnt Signaling Feed-forward Loop Promotes Proliferation Of Endogenous Neural Stem Cells After Spinal Cord Ischemia–reperfusion Injury In Rats

Objective: Activation of endogenous neural stem cells(NSCs)is thought to be an important mechanism for neural repair after mechanical spinal cord injury;however,the mechanism of the proliferation of endogenous NSCs in the spinal cord after spinal cord ischemia-reperfusion injury(SCIRI)is still unclear.In the present study,we aimed to verify the potential molecular mechanism of endogenous NSCs proliferation after SCIRI.Methods: NSCs were extracted from hippocampal of fetal rats and simulated SCIRI in vitro using oxygen-glucose deprivation/reoxygenation(OGD/R).The number of cells in each group was detected by CCK-8 assay.Edu assay was performed to detect the number of proliferating cells in each group.The dual-luciferase reporter assay verified the binding of miR-872-5p(micro RNA-872-5p)and the 3’UTR region of forkhead box protein O3a(FOXO3a),and the binding of transcription factor TCF4(T-cell factor 4)and miR-872-5p promoter.Chromatin immunoprecipitation(Ch IP)identified the binding ability of TCF4 and miR-872-5p promoter.Western Blot(WB)assay was performed to detect the expression of FOXO3 a,β-catenin and TCF4.Quantitative real time polymerase chain reaction(RT-q PCR)assay was performed to detect the expression of miR-872-5p and FOXO3 a m RNA in each group.A rat SCIRI model was established and the proliferation of endogenous NSCs was observed by immunofluorescence assay.Basso Beattie Bresnahan(BBB)score,inclined plane assay and footprint analysis were used to assess the hindlimb motor function of rats.Results: Proliferation of NSCs was observed after OGD/R in vitro.WB experiments and RT-q PCR experiments showed that FOXO3 a was reduced in NSCs after OGD/R and promoted NSCs proliferation.Mi R-872-5p increased the protein expression of β-catenin and TCF4 by targeting FOXO3 a.The results of CCK-8 and Edu assays showed that miR-872-5p promoted the proliferation of NSCs by targeting FOXO3 a.In addition,the results of dual-luciferase reporter assay and Ch IP-q PCR showed that TCF4 acted as a transcription factor to increase the expression level of miR-872-5p and that knockdown of FOXO3 a enhanced the binding of TCF4 to the miR-872-5p promoter.Immunofluorescence results showed an increased percentage of Nestin+Brd U(5-bromo-2’-deoxyuridine)positive cells in rat spinal cord after intrathecal injection of miR-872-5p.Moreover,the motor function of the hind limbs of the rats was partially restored.Conclusions: In vitro OGD/R stimulated the miR-872-5p/FOXO3a/β-catenin-TCF4 pathway,thereby promoting the proliferation of NSCs.In addition,FOXO3 a affects the binding of TCF4 transcription factor to miR-872-5p,forming a positive feedback loop that promotes the proliferation of NSCs.Mi R-872-5p promoted the proliferation of endogenous NSCs and the recovery of hind limb motor function in SCIRI rats.