The Effects And Mechanisms Of MicroRNA-132-3p,Dexamethasone And NS1619 On The Permeability Of The Blood-brain Tumor Barrier

Glioma is the most common tumors in the central nervous system(CNS).The therapies for glioma primarily include surgery,radiotherapy and chemotherapy,while the median survival time of patients less than 14.6 months[1].Blood-brain tumor barrier(BTB)impedes anti-tumor drug into the brain tumor tissues[2-4].Therefore,the improvement in BTB permeability is a necessary precondition for the treatment of glioma.Firstly,we studied the mechanisms and effects of microRNA-132-3p(miR-132-3p)on the bjood-brain tumor barrier(BTB)permeability.The immortalized human brain endothelial cell line hCMEC/D3 and the human glioblastoma cell line U87-MG were cocultured by transwell to establish the BTB models in vitro,and the hCMEC/D3 cells in this model were refered to as glioma endothelial cells(GECs).Compared with the normal endothelial cells(NECs),miR-132-3p was overexpressed in GECs,while the PTEN protein expression was at a lower level MiR-132-3p complementary base-pairing with the PTEN mRNA 3’untranslated regions(3’UTR)sequences,which will lead to the decrease of PTEN protein expression,was confirmed by the bio informatics database retrieved results and the dual-lciferase reporter system results.The vesicles of GECs stained by Alexa 488 conjugated cholera toxin B subunit(AF488-CTB)was increased by the overexpression of miR-132-3p,which illustrated that miR-132-3p improved the permeability of transcellular pathway in BTB model.On the other hand,miR-132-3p had little effect on the permeability of paracellular pathway owning to the almost unchanged transepithellal electric resistance(TEER)and tight junction proteins expression.In this study,miR-132-3p/PTEN/PKB/SRC/Caveolin-1 pathway was verified by western blot and immunocytochemistry(ICC)thoroughly combined with the PTEN overexpression vectors and specific inhibitors of PI3K or SRC.Afterwards,the findings above were conformed by AMC osmosis tests in vivo and in vitro,and the results revealed that miR-132-3p induces the increase of the BTB permeability.From the above statements,we concluded that miR-132-3p complementarily base-pairs with the PTEN 3’UTR,and lead to the decrease of PTEN protein,which will activate the miR-132-3p/PTEN/PKB/SRC/Caveolin-1 pathway.Phosphorylated Caveolin-1as the marker and functional protein of vesicles,can mirror the amount and function of vesicles in GECs.That is to say,miR-132-3p can increase the BTB permeability by transcellular ways.Secondly,we investigated the effects of dexamethasone(DEX)on the blood-brain tumor barrier(BTB)permeability and its potential mechanism.AF488-CTB marked GECs vesicles illustrated that DEX increased the amounts of vesicles in GECs,and the realtime PCR results showed that DEX upregulated the expression of miR-132-3p in GECs.Previous study expounded that miR-132-3p/PTEN/PKB/SRC/Caveolin-1 pathway increase the BTB permeability by transcellular ways.In this study,DEX reduced the expression of PTEN,and actived the downstream PKB/SRC/Caveolin-1 which led to the increase of GECs vesicles.AMC osmosis test revalidated that DEX could increase the osmosis of AMC in BTB model Glioma rat tumor AMC retention test showed that DEX could upregulate the AMC retention in glioma.In conclusion,DEX increased the expression of miR-132-3p in GECs,and led to the activation of PTEN/PKB/SRC/Caveolin-lpathway,ultimately came to the upregulation of BTB permeability.Finaly,we researched the the mechanism of a calcium-activated potassium channel(Kca channel)activator-NS1619 in the blood-brain tumor barrier(BTB)permeability.Using the rat brain glioma(C6)model,the expression of Caveolin-1,FoxO1and pFoxO1 were examined at different time points after intracarotid infusion of NS 1619 at a dose of 0.9 mg/kg.Internalization of Cholera toxin subunit(CTB)labeled fluorescence was monitored by flow cytometry.The expression of Caveolin-1 and FoxO1 at tumor micro vessels was enhanced and caveolae-mediated CTB endocytosis was increased by NS 1619 infusion for 15 min.Compared with the 15 min group,the expression of Caveolin-1 was significantly decreased and the level of phosphorylation of FoxO1 was significantly increased in the NS1619 2 h group.In addition,inhibitors of reactive oxygen species(ROS)or PI3K or PKB significantly attenuated the level of FoxO1phosphorylation and also increased the expression of caveo lin-1 protein in hCMEC/D3cocultured with U87-MG 2 h after NS 1619 treatment This led to the conclusion that NS1619-mediated transport vesicle increase is related to the ROS/P13K/PKB/FoxO1 signaling pathway.In conclusion,the studies demonstrated the both miR-132-3p/PTEN/PKB/SRC/Caveolin-1 and the ROS/PIK/PKB/FoxO1/Caveolin-1 signal pathways could be involved in increasing the permeability of BTB,and provided the experimental evidences for the clinic therapy of glioma.