Biosynthetic CircRNA₀01160 Induced By PTBP1 Regulates The Permeability Of Btb Via The CircRNA₀01160/miR-195-5p/ETV1 Axis

Objective: Glioma is the most common and deadly type of primary malignant brain tumor.Due to the presence of the blood tumor barrier?BTB?,the transportation of anti-tumor drugs into the central nervous system is impeded,which affects the effectiveness of targeted treatment of drugs.Therefore,the selective opening of BTB has become a hot spot for the treatment of glioma.The purpose of this study is to elucidate the expression and function of PTBP1,circRNA₀01160,miR-195-5p and ETV1 in human glioma brain microvascular endothelial cells in order to clarify the possible intermolecular modes of action,the molecular mechanisms regulating BTB permeability,and provide a new direction for the comprehensive treatment of glioma.Methods: In this experiment,in vitro BBB and BTB barrier models were prepared according to accepted methods,and astrocytes and glioma microvascular endothelial cells?GECs?were obtained.QRT-PCR was used to detect the expression levels of PTBP1,circRNA₀01160,miR-195-5p and ETV1.Western blot was used to detect the expression levels of PTBP1,ETV1,ZO-1,occludin and claudin-5.Stable transfection of PTBP1 silencing plasmid,circRNA₀01160 silencing plasmid,ETV1 silencing and overexpression plasmid,transient transfection of miR-195-5p silencing and overexpression plasmid,corresponding empty vector NC plasmid in human brain microvascular endothelial cells,and prepareed BTB model in vitro.The TEER value,HRP exudation and cellular immunofluorescence experiments were used to detect the mechanism of the above factors in regulating BTB permeability.The Nascent RIP experiment was used to detect the interaction between PTBP1 and ANKRD17pre-m RNA.The dual luciferase reporter gene assay was used to analyze the targeted binding effect and binding site of circRNA₀01160 to miR-195-5p and miR-195-5p to ETV1 3'-UTR region.The luciferase reporting experiment and CHIP experiment were used to detect the interaction of ETV1 with the tight junction related proteins ZO-1,occludin,and claudin-5 promoter regions.Apoptosis experiments were used to detect the effects of transfection of silenced PTBP1,silenced circRNA₀01160,overexpression of miR-195-5p and doxorubicin on the apoptosis rate of U87 cells in an in vitro BTB model.Results: This study found that PTBP1 was highly expressed in GECs,and silencing PTBP1 could significantly increase BTB permeability.CircRNA₀01160 was highly expressed in GECs,and silencing circRNA₀01160 significantly increased BTB permeability.PTBP1 could induce the production of circRNA₀01160,and double-silencing PTBP1 and circRNA₀01160 could evidently increase BTB permeability.MiR-195-5p was underexpressed in GECs,and overexpression of miR-195-5p distinctly increased BTB permeability.CircRNA₀01160 and miR-195-5p had a targeted binding effect.CircRNA₀01160 mitigated its inhibitory effect on the target gene ETV1 by adsorbing miR-195-5p.ETV1 was highly expressed in GECs,and overexpression of ETV1 could significantly increase the expression of tight junction-associated proteins and reduce BTB permeability.MiR-195-5p bound to the3'-UTR region of ETV1 mRNA.Double overexpression of miR-195-5p and ETV1 significantly reversed the effect of miR-195-5p overexpression alone on BTB permeability.ETV1 bound to the promoter regions of tight junction-associated proteins ZO-1,occludin,and claudin-5,and visibly promoted its expression at the transcription level.In the BTB model in vitro,PTBP1 silencing,circRNA₀01160 silencing,and overexpression of miR-195-5p combined with doxorubicin markedly promoted glioma cell apoptosis.Conclusions: 1.In human glioma microvascular endothelial cells,PTBP1,circRNA₀01160 and ETV1 were highly expressed,and miR-195-5p was lowly expressed.2.PTBP1 affected the generation of circRNA₀01160.By adsorbing miR-195-5p,circRNA₀01160 attenuated the negative regulatory effect of miR-195-5p on its target gene ETV1,thereby regulating BTB permeability.3.Silencing PTBP1,silencing circRNA₀01160 and the combined application of overexpressed miR-195-5p and doxorubicin could significantly promote the apoptosis of glioma cell U87.4.PTBP1 played an important role in regulating BTB permeability through the CircRNA₀01160 /miR-195-5p / ETV1 axis,and targeting PTBP1 / CircRNA₀01160 / miR-195-5p / ETV1 axis will provide treatment for glioma effective target,safer and more effective treatment of glioma.