The Mechanism Of Low-dose Endothelial -Monocyte Activating Polypeptide-? Increase The Permeability Of Blood Tumor Barrier Via MiR-330-3p/PKC-? Signaling Pathway

Objective:In the glioma,blood-tumor barrier?BTB?is the key factor to restrict the delivery of large therapeutic molecules into the tumor tissues,which affects the efficacy of chemotherapy.BTB has different characteristics from the BBB,which results from damage caused by glioma cells development.Although the permeability of BTB is slightly higher than BBB,BTB still poses a major hurdle to anticancer drug delivery to tumors.Therefore,selectively enhance the permeability of BTB is an urgent problem to be solved to delivery anti-tumor drugs into tumor tissues effectively and to improve the therapeutic effect of glioma.Endothelial monocyte-activating polypeptide-??EMAP-??,which is synthesized by its precursor proEMAP-?,can regulate the function of endothelial cells and monocytes.At present,it has been found that EMAP-? has many functions of regulating cell function.Studies have reported that low dose EMAP-??0.05nM?selectively increases the permeability of BTB via the cAMP/PKA signaling pathway and the PKC-?/PP2A signaling pathway.In vitro BTB model,low-dose EMAP-? can bind to?-ATP synthase on BMECs surface and open tight junction?TJ?to selectively increase the permeability of BTB by significantly decreasing the protein expression levels of TJ-related proteins ZO-1,occludin and claudin-5.Other studies have demonstrated that protein kinase C?PKC?has distinct effects on the dynamic changes in TJ and the permeability of endothelial cells.PKC activation resulted in phosphorylation and redistribution of TJ related proteins,contributes to regulate the TJ^[12].It was found that EMAP-? can increase the permeability of BTB model in vitro by activating PKC,the effects of EMAP-? on BTB permeability were significantly diminished by H7,the inhibitor of PKC.The above studies suggest that PKC plays an important regulatory role in EMAP-? increases the permeability of BTB.However,the mechanism of EMAP-II regulating the expression and activity of PKC is unclear.MicroRNAs are a class of small non-coding RNAs with18-25 nucleotides in length.MiRNAs exert effects on regulating target genes by post-transcriptional,and regulates the biological functions of many kinds of cells in physiological and pathological processes.MircoRNA-330?miR-330?gene was discovered by Weber in 2005,located in 19q13.32,expressed in a variety of tissues with different functions.MiR-330-3p,as one of the important subtypes of miR-330,plays different roles in different tissues.However,the effect of miR-330-3p on brain microvascular endothelial cells of glioma has not yet been reported.Potential binding site between the 3'-untranslated region?UTR?of PKC--?mRNA and the seed region of miR-330-3p was predicted.Bioinformatics analyses reveal two conseved target sites for miR-330-3p in the mRNA of PKC-?3'UTR at nucleotides 2038-2044 and 3115-3121.Therefore,we hypothesized that mi R-330-3p is likely involved in the regulation of PKC-?by which EMAP-? increase BTB permeability.This study aims to investigate the endogenous expression of mi R-330-3p in glioma microvascular endothelial cells and whether the expression of miR-330-3p is regulated by EMAP-?.Further study whether EMAP-II affects the permeability of BTB by regulating the expression of miR-330-3p and the effect and possible mechanism of miR-330-3p regulating BTB permeability.In this study,we aim to explore the molecular mechanisms associated with low-dose EMAP-II selectively increasing the permeability of BTB and provide new ideas for comprehensive treatment of glioma.Methods:1.Cell cultures of human cerebral microvascular endothelial cell line hCMEC/D3,human glioma cell line U87 and human embryonic kidney cell line HEK293T.2.Establishment of an in vitro BTB model.3.Celltransfection and generation of stable expressing cells:miR-330-3p over-expression,miR-330-3p silencing,stable expressing hCMEC/D3 and U87.4.The expression levels of miR-330-3p were detected by real-time PCR.5.The permeability of BTB model was evaluated by transendothelial electric resistance?TEER?.6.The permeability of BTB model was evaluated by Horseradish peroxidase?HRP?flux.7.The protein expression of tight junction related proteins ZO-1,occludin and claudin-5 in hCMEC/D3 were evaluated by Western blot and real-time PCR.8.The protein expression of PKC-?in hCMEC/D3 were evaluated by real-time PCR,Western blot and laser scanning confocal microscope assays.9.Regulation of miR-330-3p targeting PKC-?3'UTR were evaluated by Dual luciferase reporter gene assay.10.The protein expression of tight junction related proteins ZO-1,occludin and claudin-5 in hCMEC/D3 were evaluated by Immunofluorescence assays.11.Apoptosis was evaluated to compare the effects of DOX,EMAP-?+anti-miR-330-3p+PKC-?activator and EMAP-?+anti-mi R-330-3p+PKC-?activator+DOX on cell viability and apoptosis in U87 glioma cells.Results:1.miR-330-3p expression was up-regulated in GECs.2.MiR-330-3p expression was down-regulated in GECs by low dose EMAP-II,in 1h,the expression is the lowest.3.MiR-330-3p over-expression significantly decreased the permeability of the BTB,miR-330-3p silencing significantly increased the permeability of the BTB,EMAP-? treatment induced a decrease in TEER value of the BTB as well as an increasing in HRP flux across the BTB.In BTB of miR-330-3p silencing+EMAP-?,the change was more significant.4.The over-expression of miR-330-3p increased the expression of tight junction related proteins in GECs.EMAP-? treatment induced a decrease in the expression of tight junction related proteins in GECs.In group of miR-330-3p silencing+EMAP-?,the change was more significant.5.The over-expression of miR-330-3p could downregulate the mRNA and protein expressions of PKC-?in GECs,EMAP-? treatment induced a increase of the mRNA and expression of PKC-?in GECs.In GECs of mi R-330-3p silencing+EMAP-?,the change was more significant.6.Observed by the laser scanning confocal microscope,in the over-expression group,the immunoreactivity of PKC-?was weakened,EMAP-II treatment induced a increasing.In mi R-330-3p silencing+EMAP-? group,the immunoreactivity of PKC-?was enhanced significantly.7.MiR-330-3p inhibited the expression of PKC-?by targeting its 3?-UTR.8.BTB permeability and expression of tight junction-related proteins were affected by PKC-?specific activity or inhibitor in miR-330-3p overexpressed or silencing cells.Conclusions:1.miR-330-3p expression was up-regulated in GECs.Over-expression of miR-330-3p could decreased the permeability of BTB,and increase the expression of ZO-1,occludin and claudin-5 in GECs.2.MiR-330-3p inhibited the expression of PKC-?by targeting its 3?-UTR and regulated BTB permeability through the alteration of PKC-?activity.3.Low dose EMAP-? increase permeability of BTB via downregulating mi R-330-3p,then increasing the expression and activity of PKC-?,increasing the expression of ZO-1,occludin and claudin-5.4.The combination of EMAP-?,anti-mi R-330-3p and PKC-?activator significantly enhanced the antitumor effects of DOX on the glioma cells.