| Objective: Alzheimer's disease is a central nervous system degenerative disease characterized by progressive cognitive dysfunction and behavioral impairment.It is the most common type of dementia and is further increased as the population ages.Existing treatments only moderately improve symptoms but cannot be cured.Alzheimer's disease not only causes serious pain to patients and caregivers,but also imposes a huge economic burden on society.The pathogenesis of alzheimer's disease is complex,mainly involving abnormal metabolism of ?-amyloid,hyperphosphorylation of tau protein,oxidative stress,and damage of blood-brain barrier.The blood-brain barrier(BBB)can restrict neurotoxic substances and inflammatory factors entering the central nervous system from the periphery,and maintaining CNS homeostasis.BBB is composed of brain microvascular endothelial cells,pericytes,extracellular matrices and the foot processes of perivascular astrocytes.Moreover,tight junctions(TJs)between brain microvascular endothelial cells play critical roles in maintaining the integrity and permeability of the BBB.Occludin and claudins are intact membrane proteins that are localized between the tight junctions of brain microvascular endothelial cells.The zonula occludens-1(ZO-1)act as a scaffold connecting TJ strands woth the actin cytoskeleton of brain endothelila cells.Therefore,targeting the regulation of permeability of the blood-brain barrier may be an effective means of treating alzheimer's disease.Memantine(MEM) is a low affinity,uncompetitive glutamatergic N-methyl-D-aspartate(NMDA)receptor antagonist.It has been demonstrated that memantine could attenuates SAH-induced neurological impairment by improving impaired the permeability of BBB,inhibiting nNOS activity and peroxynitrite formation and subsequently suppressing apoptotic cascade.However,the mechanism of MEM on BBB permeability in AD microenvironment remains unclear.Long non-coding RNAs participate in biological functions at the transcriptional and post-transcriptional levels,and their roles in the central nervous system are increasingly attracting researchers' attention.SNHG1 has been dysregulated in the early stage of Parkinson's disease,which promotes the occurrence of Parkinson's neuroinflammation.SCAMP1 is widely expressed in brain tissue and is associated with synapse formation.The role of LINC00094,SNHG1 and SCAMP1 in alzheimer's disease has not been reported yet.RNA-binding proteins play important roles in the regulation of post-transcriptional levels,including RNA splicing,RNA transport,and mRNA stabilization.Studies have shown that RNA-binding proteins can bind to non-coding RNAs to affect their function.MOV10 was overexpressed in cervical cancer.However it has not been reported in Alzheimer's disease.MiRNAs are involved in a variety of cellular processes such as cell proliferation,differentiation,apoptosis,stress response and metabolism,and their role in neurodegenerative diseases has been widely reported.At present,the regulation of miR-224-5p and miR-497-5p on the permeability of blood-brain barrier has not been reported.Our study firstly demonstrated the expression and the function of LINC00094,MOV10 and SNHG1/SCAMP1 in Abeta-incubated human brain microvascular endothelial cells,and further explored the possible regulatory mechanisms between these factors and the effect on blood-brain barrier permeability of alzheimer's disease.The present study aims to provide new strategy for the treatment of alzheimer's disease from the perapective of the blood brain barrier.Methods: 1.The establishment of BBB model in vitro.2.The expression levels of LINC00094,miR-224-5p,miR-497-5p,Endophilin-1,MOV10,SNHG1,SCAMP1,FOSL2 and NFE2 were determined by real-time PCR.3.The expression levels of Endophilin-1,ZO-1,occludin,claudin-5,MOV10,FOSL2 and NFE2 were determined by Western blot.4.Abeta-incubated endothelial cells were stably transfected with LINC00094 knockdown,Endophilin1-1 overexpression,MOV10 knockdown,SNHG1 knockdown,SCAMP1 knockdown,FOSL2 overexpression,FOSL2 knockdown,NFE2 overexpression,NFE2 knockdown plasmids,and were transiently transfected with agomir-224-5p/agomir-497-5p and antagomir-224-5p/antagomir-497-5p.5.The BBB integrity was determined by Transendothelial electric resistance(TEER)assay.6.The BBB permeability was determined by horseradish peroxidase(HRP)flux assay.7.The expression and distribution of tight junction-associated proteins ZO-1,occludin and claudin-5 were detected by immunofluorescence assay.8.The binding sites of LINC00094 and miR-224-5p/miR-497-5p,miR-224-5p/miR-497-5p and Endophilin-1 were determined by dual-luciferase reporter assay.9.The binding of LINC00094,and miR-224-5p/miR-497-5p with Ago2,the binding of MOV10 with SNHG1 and SCAMP1,the binding of SNHG1 and SCAMP1 with EZH2 were determined by RNA binding protein immunoprecipitation(RIP)assay.10.The association of FOSL2/NFE2 and the promoter regions of ZnT1,ZnT3 and MOV10 were determined by Chromatin immunoprecipitation(ChIP).Results: 1.LINC00094 was upregulated in AD-ECs and downregualted in MEM-ECs.Knockdown of LINC00094 significantly increased the TEER values and decreased the HRP flux.Meanwhile knockdown of LINC00094 significantly promoted the expression of ZO-1,occludin and claudin-5.Furthermore,the distribution of ZO-1,occludin and claudin-5 became continues on cell boundaries following LINC00094 knockdown.2.The combination of MEM and knockdown LINC00094 has the most obvious effect of reducing BBB permeability.3.Meanwhile LINC00094 targeted miR-224-5p/miR-497-5p,and were enriched in Ago2 immunoprecipitates with miR-224-5p/miR-497-5p.4 Overexpression of miR-224-5p/miR-497-5p in AD-ECs that stably knocked down LINC00094 significantly promoted the increases in TEER values,the decreases in HRP flux by LINC00094 knockdown alone,as well as promoted the expression of ZO-1,occludin and claudin-5.On the contrary,downregulation of miR-224-5p/miR-497-5p in AD-ECs that stably knocked down LINC00094 significantly revised the above effects.5.miR-224-5p/miR-497-5p was downregulated in GECs.MiR-224-5p/miR-497-5p overexpression significantly increased the TEER values and decreased the HRP flux..Furthermore,the distribution of ZO-1,occludin and claudin-5 became continues on cell boundaries following miR-224-5p/miR-497-5p overexpression.6.miR-224-5p/miR-497-5p targeted Endophilin-1.Meanwhile,miR-224-5p/miR-497-5p overexpression significantly inhibited Endophilin-1 expression in AD-ECs.7.Co-overexpression of Endophilin-1 and miR-224-5p/miR-497-5p in AD-ECs,significantly revised the increases in TEER value,the decreases in HRP flux by miR-224-5p/miR-497-5p overexpression alone,as well as promoted the ZO-1,occludin and claudin-5 expression.8.MOV10 was upregulated in AD-ECs and MOV10 inhibition significantly reduced the BBB permeability in AD microenvironment.9.SNHG1 and SCAMP1 were elevated in AD-ECs and silencing of SNHG1 and SCAMP1 increased the BBB permeability.10.Knockdown of MOV10 stabilized SNHG1,SCAMP1 and upregulated SNHG1,SCAMP1 in AD-ECs.11.FOSL2 and NFE2 are upregulated in AD-ECs,and knockdown of FOSL2 and NFE2 reduced BBB permeability.12.SNHG1 and SCAMP1 inhibitd the expression of FOSL2 and NFE2 via the PRC2 complex pathway.13.FOSL2/NFE2 bound to the promoter regions of ZnT1,ZnT3 and MOV10 and promotes their expression,forming a feedback loop affecting the permeability of BBB in AD microenvironment.Conclusion: 1.MEM combined with knockdown of LINC00094 to reduce BBB permeability.2.LINC00094 attenuates post-transcriptional inhibition of Endophilin-1 via binding to miR-224-5p/miR-497-5p to regulate BBB permeability.3.MEM regulates BBB permeability in the AD microenvironment via the LINC00094/miR-224-5p(miR-497-5p)/Endophilin-1 axis.4.MOV10,FOSL2 and NFE2 were upregulated in AD-ECs.Silencing MOV10,FOSL2 and NFE2 respectively,reduced the permeability of BBB in AD microenvironment.SNHG1 and SCAMP1 are downregulated in AD-ECs,and knockdown of SNHG1 and SCAMP1 increased the permeability of BBB in AD microenvironment.5.MOV10 reduced BBB permeability in AD microenvironment via decreasing the stability of SNHG1/SCAMP1.SNHG1/SCAMP1 regulates the expression of FOSL2/NFE2 through the PRC2 complex pathway and affects the BBB permeability in AD microenvironment.6.MOV10 combined with SNHG1/SCAMP1 to form a feedback loop to regulate the permeability of BBB in AD microenvironment. |
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