| Objective: Micro RNA(mi RNA)is closely related to the formation of brain edema,but its mechanisms is not clear.This study aims to investigate the molecular mechanisms of whether mi R-18 a decreases the tight junction associated protein Occludin and ZO-1 expressions via regulating RUNX1 after intracerebral hemorrhage(ICH),resulting in BBB permeability damage and form perihematomal brain edema.Methods: 1.Cell experiment in vitro: The BBB cell model of brain microvascular endothelial cells(BMVEC)co-cultured with astrocytes(AC)and the BBB cell model of ICH pretreated with thrombin were established in vitro.The expressions of BMVEC and AC were detected with factor VIII and GFAP by immunofluorescence.BBB cell model were divided into eight groups:(1)model group:After the BBB cell model was established,20U/m L thrombin was added and cultured for 24 h to construct the BBB cell model of intracerebral hemorrhage in vitro;(2)overexpression control group: the control adenovirus(empty vector)with mi R-18 a overexpression was added at the ratio of MOI=1:10(Multiplicity of Infection,the ratio of the virus to the number of cells);(3)mi R-18 a overexpression group(mi R-18a): adenoviruses that overexpress mi R-18 a were added at the ratio of MOI=1:10(viruses can count 10 times to the number of cells);(4)interference control group: mi R-18 a interference control adenovirus(empty vector)was added at the ratio of MOI=1:10;(5)mi R-18 a interference group(sh-mir-18a): adenovirus interfering with mi R-18 a was added at the ratio of MOI=1:10;(6)normal control group: RUNX1 overexpression control adenovirus(empty vector)was added at the ratio of MOI=1:10;(7)mi R-18 a and RUNX1 were both overexpressed groups(mi R-18 a +RUNX1):adenoviruses which expressed mi R-18 a and RUNX1 were added at a ratio of MOI=1:10,respectively;(8)mi R-18 a and RUNX1 co-interfering groups(sh-mir-18a+sh-runx1): adenoviruses which interfered with mi R-18 a and RUNX1 were added at the ratio of MOI=1:10,respectively.mi R-18 a or RUNX1 was up or down-regulated by adenovirus transfection.The BBB permeability in vitro was evaluated by transendothelial electrical resistance(TEER)and fluorescein sodium(Na-Flu).The expressions of mi R-18 a,RUNX1,Occludin and ZO-1 were detected by q RT-PCR and Western Blot,and the binding of mi R-18 a to RUNX1 was verified by dual-luciferase reporter assay.2.Animal experiments in vivo: SD rats model for ICH was established,It was divided into six groups:(1)sham group(sham group,as the control group,simulating the modeling process of intracerebral hemorrhage in rats,without blood injection,);(2)intracerebral hemorrhage model group(model group,intracerebral hemorrhage model was prepared by injecting 50?L of autogenous non-anticoagulant arterial blood into basal ganglia region in rats);(3)mi R-18 a overexpression group(mi R-18 a group,1?L of adenovirus overexpression of mi R-18 a were injected at 4 sites around the hematoma in rats with intracerebral hemorrhage model);(4)mi R-18 a interference group(sh-mi R-18 a group,1?L of adenovirus interfere with mi R-18 a were injected at 4 sites around the hematoma in rats with intracerebral hemorrhage model);(5)mi R-18 a overexpression + RUNX1 overexpression group(adenovirus 2?L with overexpression of mi R-18 a and RUNX1 were injected at 4 sites around the hematoma in rats with intracerebral hemorrhage model,and mixed virus before injection);(6)mi R-18 a +RUNX1interference group(sh-mir-18a+sh-runx1 interference group,adenovirus 2?L with interference with mi R-18 a and RUNX1 were injected at 4 sites around the hematoma in rats with intracerebral hemorrhage model,and mixed virus before injection),and each of the above six groups of rats was further divided into 3subgroups of 3d,5d and 7d,10 rats in each group,n=30 and mi R-18 a or RUNX1 was up or down-regulated by adenovirus transfection,q RT-PCR and Western Blot were used to detect the expressions of mi R-18 a,RUNX1,Occludin and ZO-1,and immunofluorescence staining was used to observe the expressions of RUNX1,Occludin and ZO-1 proteins.Meanwhile,the changes of neurological deficit score(Longa score),BBB permeability and brain water content were observed.The binding of RUNX1 with the Occludin and ZO-1 gene promoter regions was observed by chromatin immunoprecipitation(Ch IP)technique.Results: 1.Cell experiment in vitro:(1)BBB cell model in vitro which co-cultured with BMVEC and AC was established and identified by immunofluorescence with factor VIII and GFAP successfully.(2)Overexpression of mi R-18 a led to a decrease in TEER,and led to an increase in Na-Flu when compared with the model group in vitro(P<0.05).On the contrary,interference with mi R-18 a give raise to an increase in TEER,and give raise to a decrease in Na-Flu when compared with the model group(P<0.05).(3)Both q RT-PCR and Western Blot indicated that up-regulation of mi R-18 a rusult in a decrease in RUNX1,Occludin and ZO-1,while down-regulation of mi R-18 a give raise to an increase expressions of the above three proteins when compared with the model group(P<0.05).(4)Dual-luciferase reporter assay showed that relative luciferase activity was decreased in mi R-18a+RUNX1 wild-type plasmid group when compared with the control+RUNX1 wild-type plasmid group(P<0.05),indicating that mi R-18 a can combine with RUNX1 gene promoter,inhibit wild-type plasmid luciferase activity effectively,relative luciferase activity was not changed in mi R-18a+RUNX1 mutant-type plasmid group when compared with the control +RUNX1 mutant-type plasmid group(P>0.05),indicating that mi R-18 a can not combine with RUNX1 mutant-type plasmid.mi R-18 a can combine with RUNX1,the target gene of mi R-18 a is RUNX1.2.Animal experiments in vivo:(1)The ICH model in SD rats were established successfully.(2)Both q RT-PCR and Western Blot indicated that overexpression of mi R-18 a caused a decrease in RUNX1,Occludin and ZO-1,while interference with mi R-18 a resulted in an increase in the expressions of the above three proteins when compared with the model group(P<0.05).(3)Immunofluorescence staining showed that overexpression of mi R-18 a resulted in a decreased expressions of RUNX1,Occludin and ZO-1 proteins,while down-regulation of mi R-18 a resulted in an increased expressions of the above three proteins when compared with the model group(P<0.05).(4)Langa score reached the peak in the3 day group,decreased slightly in the 5day group,and the 7day group had the lowest Longa score.Overexpression of mir-18 a resulted in an increased Longa scores in rats,while down-regulation of mi R-18 a resulted in a decreased Longa scores when compared with the model group(P<0.05).These results showed that the nerve function was improved via interfering with mi R-18 a.(5)For BBB permeability,overexpression of mi R-18 a leads to an increased BBB permeability,while interference with mi R-18 a resulted in a decreased BBB permeability when compared with the model group(P<0.05).(6)The perihematomal brain water content was increased via mi R-18 a up-regulation and decreased via mi R-18 a down-regulation when compared with the model group(P<0.05).(7)Ch IP-q PCR experiment showed that overexpression of mi R-18 a inhibited the binding of RUNX1 with Occludlin and ZO-1 promoter,and interfered with mi R-18 a promoted the binding of RUNX1 with Occludlin and ZO-1 promoter when compared with the model group(P<0.05).Conclusion: 1.The expressions of mi R-18 a and RUNX1 showed the opposite trend,the mi R-18 a increased and the RUNX1 decreased.2mi R-18 a can bind to the 3'UTR of RUNX1 m RNA.RUNX1 is the target gene of mi R-18 a,and Occludin and ZO-1 are the target proteins regulated by RUNX1.mi R-18 a can inhibit the expressions of Occludin and ZO-1 proteins via regulating the expression of RUNX1,increase BBB permeability and aggravate brain edema.3.Blocking of mi R-18 a /RUNX1 pathway,the expressions of Occludin and ZO-1,BBB permeability and brain edema decreased,the neurological function of rats with intracerebral hemorrhage was improved.4.At different time points on the 3d,5d and 7d after intracerebral hemorrhage,the expressions of mi R-18 a,RUNX1,Occludin and ZO-1 were different,the destruction of BBB and the severity of brain edema were different,and the BBB permeability reached a peak after intracerebral hemorrhage in 3d group. |
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