Effects And Mechanisms Of MiRNA-429 Regulating Kruppel-like Factor 6 On Structure And Function Of Blood Spinal Cord Barrier

ObjectiveBy establishing an in vitro blood-spinal cord barrier(BSCB)model,we investigated the effect of miRNA-429(miR-429)targeting Kruppel like factor 6(KLF6)on the permeability of the blood-spinal cord barrier by regulating tight junction protein transcription.MethodsTransfected antagomiR-429(silent group),antagomiR-429 NC,agomiR-429(enhanced group),agomiR-429 NC into immortalized human brain microvascular endothelial cells(h CMEC/D3),and then antagomiR-429,AntagomiR-429 NC,agomiR-429,agomiR-429 NC with control and KLF6(+),KLF6(+)NC,KLF6(-),KLF6(-)NC co-transfected into immortalized Brain microvascular endothelial cells(h CMEC / D3)are divided into: control group,agomiR-429 NC + KLF6(+)NC group,agomiR-429 + KLF6(+)group,agomiR-429 NC + KLF6(-)NC group,agomiR-429 + KLF6(-)group,antagomiR-429 NC + KLF6(+)NC group,antagomiR-429 + KLF6(+)group,antagomiR-429 NC + KLF6(-)NC group,antagomiR-429 + KLF6(-)group.h CMEC / D3 and spinal astrocytes(Ha-sc)were used to construct an in vitro BSCB model on a Transwells filter.RT-q PCR,Western blot,and immunofluorescence were used to detect the expression of Krüppel-like factor 6(KLF6)protein and three TJ proteins: ZO-1,Occludin,and Claudin-5.The permeability of BSCB in vitro was determined by HRP flux method.ResultsThe relative expression of KLF6 after transfection of agomiR-429 was significantly lower than that of the control group and NC group(P<0.05),and the relative expression of KLF6 after transfection of antagomiR-429 was significantly higher than that of the control group and NC group(P<0.05).The relative expression levels of ZO-1,Occludin,and Claudin-5 in agomiR-429 group were significantly lower than those in control group and NC group(P<0.05).HRP penetration rate in agomiR-429 group was higher than that in control group and NC group(P<0.05);the relative expression levels of ZO-1,Occludin,and Claudin-5 in the antagomiR-429 group were significantly higher than those in the control group and the NC group(P<0.05).HRP permeability was lower than that in the control group and the NC group(P<0.05).The relative expression levels of ZO-1,Occludin,and Claudin-5 in agomiR-429 + KLF6(-)group were significantly lower than in agomiR-429 + KLF6(+)group(P<0.05),agomiR-429 + KLF6 The HRP permeability of(-)group was significantly higher than that of agomiR-429 + KLF6(+)group(P<0.05).The relative expression levels of the three TJ proteins in the antagomiR-429 + KLF6(+)group were significantly higher than those in the antagomiR-429 + KLF6(+)group(P<0.05),and the antagomiR-429 + KLF6 The HRP permeability of(+)group was significantly lower than that of antagomiR-429 + KLF6(-)group(P<0.05).ConclusionsmiR-429 targets KLF6 to regulate the tight junction protein transcription and thus affect the permeability of the blood spinal cord barrier.Provide new targets and ideas for the treatment of SCI and its mechanism.