The Effect And Mechanism Of KLF11 Regulated By MiRNA-138-5p On Blood Spinal Cord Barrier Permeability

ObjectiveBy investigating the effect and mechanism of KLF11 regulated by miRNA-138-5p on blood spinal cord barrier permeability,this study may provide new targets and ideas for the treatment of SCI and its therapeutic mechanism.MethodThe following plasmids were constructed: pMIR-KLF11 3’UTR-wt,pMIR-KLF11 3’UTR-mut,agomiR-138-5p and agomiR-138-5pNC vectors and transfected into Hep G2 cells respectively.antagomiR-138-5p,antagomiR-138-5pNC,agomiR-138-5p,agomiR-138-5pNC were translated-indirectly into h CMEC/D3 cells,Lentiviral stable constructs of KLF11(+),KLF11(+)NC,KLF11(-),KLF11(-)NC,and agomiR-138-5p and agomiR-138-5pNC were co-transfected into h CMEC/D3 and grouped into control,agomiR-138-5pNC + KLF11(+)NC,agomiR-138-5p + KLF11(+)NC,agomiR-138-5pNC+ KLF11(+)NC,agomiR-138-5pNC+ KLF11(+)and agomiR-138-5p + KLF11(+)groups,respectively.The protein expression levels of KLF11,ZO-1,claudin-5 and occludin were determined by RT qPCR and Western blot;Meanwhile,the change of FITC dextran permeability of BSCB in vitro was determined.ResultsCompared with the agomiR-138-5pNC + KLF11 3’UTR-wt group,the relative luciferase activity of cells co transfected with agomiR-138-5pNC plus KLF11 3’ UTR-wt was significantly decreased(P < 0.05),whereas the fluorescence intensity of cells co-transfected with agomiR-138-5pNC+ KLF113’UTR-mut was not significantly changed(P > 0.05);The relative expression of protein and gene of KLF11 after transfection with agomiR-138-5p was decreased compared with the control and NC groups(P < 0.05),and that of KLF11 after transfection with antagomiR-138-5p was increased compared with the control and NC groups(P < 0.05).The relative protein and gene expressions of ZO-1,claudin-5 and occludin were significantly higher in the KLF11(+)lentivirus stable than in the control and NC groups(P < 0.05),and the protein and gene expressions of ZO-1,claudin-5 and occludin were significantly lower in the KLF11(-)lentivirus stable than in the control and NC groups(P < 0.05);The protein and gene expressions of ZO-1,claudin-5 and occludin in the agomiR-138-5p + KLF11(+)NC group were significantly lower than those in the control and agomiR-138-5pNC + KLF11(+)NC groups(P <0.05),and the FITC dextran penetration rate in the agomiR-138-5p + KLF11(+)NC group was significantly higher than that in the agomiR-138-5pNC + KLF11(+)NC group(P < 0.05);The protein and gene expressions of ZO-1,claudin-5and occludin in the agomiR-138-5pNC + KLF11(+)group were significantly higher than those in the control and NC groups(P < 0.05),and the FITC dextran permeability in the agomiR-138-5pNC + KLF11(+)group was significantly lower than that in the agomiR-138-5pNC + KLF11(+)NC group(P < 0.05);The protein and gene expressions of ZO-1,claudin-5 and occludin in the agomiR-138-5p + KLF11(+)group were significantly higher than those in the agomiR-138-5p+ KLF11(+)NC group(P < 0.05),and the FITC dextran penetration rate in the agomiR-138-5p + KLF11(+)group was significantly lower than that in the agomiR-138-5p + KLF11(+)NC group(P < 0.05).ConclusionmiRNA-138-5p negatively regulates the expression of KLF11 by targeting,further inhibiting the expression of tight junction protein,thus affecting the permeability of blood-spinal cord barrier,and providing new targets and ideas for the treatment of SCI.