| Objective: Glioma are one of the most lethal diseases in the brain.The current treatment regimen is a comprehensive system of surgery,chemotherapy,radiation therapy and molecular targeted therapy.The efficient and rapid entry of drugs into brain tumor tissues is the key to improving the efficacy of chemotherapy.The blood tumor barrier（BTB）is a structure composed of three microvascular subpopulations in tumor tissues.The presence of BTB will greatly reduce the drug’s Pass rate and the efficacy of chemotherapy.Therefore,how to selectively increase the permeability of BTB and make the drug pass through BTB as much as possible will become one of the more effective strategies for comprehensive treatment of glioma.Pi RNAs（Piwi-interacting RNA）are a class of non-coding RNAs of approximately24 to 32 nt（nucleotides）in length.Among all non-coding RNAs,the number of pi RNAs is the highest.pi RNAs exert biological functions by interacting with PIWI proteins.Recent studies have found that PIWI/pi RNA is closely related to the occurrence and development of diseases such as tumors,and can play an important role in the protein regulation,epigenetic regulation and transposon silencing of genes.pi RNAs may become biomarkers for future tumor diagnosis and treatment.Functional studies of pi RNA-DQ590027（hsapi R014633）have not been reported.Long non-coding RNAs（lncRNAs）are a class of non-coding RNAs that are involved in the regulation of various biological processes in cells and are between 200 and 100,000 nt in length.It plays an important regulatory role in gene expression at both transcription and post-transcriptional levels.Related studies have shown that some specific lnc RNAs will undergo abnormal changes in expression levels in tumor cells,and lnc RNAs may serve as potential drug targets for future treatment of tumor diseases as well as new diagnostic markers.MIR17 HG is a host gene encoding the mi R-17-92 gene cluster.Studies have shown that MIR17 HG is involved in the regulation of various tumors,but the role of endothelial cell function in glioma has not been reported.This study firstly explored the expression and function of pi RNA-DQ590027 and lnc RNA-MIR17 HG in human glioma microvascular endothelial cells,and further explored the possible mechanism of the above factors regulating vascular endothelial cells of glioma and explained the permeability of BTB.The regulation role provides a new idea for the comprehensive treatment of glioma in the future.Research methods: 1.Establish in vitro BTB model;2.Real-time quantitative PCR method to detect the expression levels of pi R-DQ590027,MIR17 HG,mi R-153,mi R-377 and FOXR2;3.Western bolt detection of FOXR2,ZO-1,occludin,Claudin-5 expression level;4.Immunofluorescence staining assay for ZO-1,occludin,claudin-5;5.Silencing MIR17 HG,overexpressing FOXR2,silencing FOXR2 in GECs,constructing stable MIR17 HG silencing,FOXR2 overexpression and silencing transfected cell lines and transient transfection of agomir-153 and antagomir-153,agomir-377 and antagomir-377,respectively,in GECs;6.TEER assay to detect BTB integrity;7.HRP exudation assay to detect BTB permeability;8.Dual luciferase reporter gene experiments to verify the targeting and targeting sites of pi R-DQ590027 and MIR17 HG,MIR17HG and mi R-153,MIR17 HG and mi R-377,mi R-153 and FOXR2,mi R-377 and FOXR2;9.RNA-binding protein immunoprecipitation assay（RIP）was used to detect the targeted binding of MIR17 HG,mi R-153 and mi R-377 to Ago2;10.Ch IP assay was used to verify the targeted binding of t FOXR2 to the promoter region of ZO-1,occludin and claudin-5,respectively;11.Flow cytometry technology was used to detect the application of blood tumor barriers.Results: 1.pi R-DQ590027 was lowly expressed in GECs.Overexpression of pi R-DQ590027 significantly decreased TEER value and increased HRP exudation rate.It also significantly inhibited the expression of tight junction-associated proteins ZO-1,occludin and claudin-5 in GECs and changed the distribution of tight junction-associated proteins ZO-1,occludin,and claudin-5 on the cell membrane of GECs,which changed from a relatively continuous distribution to a discontinuous distribution;silencing pi R-DQ590027 significantly increased the TEER value and decreased the exudation of HRP At the same time,the expression of tight junction-associated proteins ZO-1,occludin and claudin-5 in GECs was significantly enhanced,and the distribution of tight junction-associated proteins ZO-1,occludin and claudin-5 on the cell membrane was increased.2.MIR17 HG is highly expressed in GECs.Knockdown of MIR17 HG significantly decreased TEER value and increased HRP exudation rate.It also significantly inhibited the expression of tight junction-associated proteins ZO-1,occludin,claudin-5,and changed the distribution of the tight junction-associated proteins ZO-1,occludin,and claudin-5 from a relatively continuous state to a discontinuous state.3.Overexpression of pi R-DQ590027 in GECs significantly reduced the expression of MIR17 HG.At the same time,pi R-DQ590027 bound to MIR17 HG.4.Overexpression and silencing of pi R-DQ590027 in MIR17HG-expressing GECs,overexpression of pi R-DQ590027 significantly decreased TEER value,increased HRP exudation rate,and significantly inhibited the expression of brain tight junction-associated protein ZO-1,occludin and claudin-5;silencing pi R-DQ590027 significantly inhibited the inhibitory effect of MIR17 HG on TEER and the promotion of HRP,and significantly inhibited the underexpression of tight junction-associated proteins ZO-1,occludin and claudin after silencing MIR17 HG.5.mi R-153 is underexpressed in GECs.Overexpression of mi R-153 can significantly reduce TEER value,increase HRP exudation rate,and significantly inhibit the protein expression of tight junction-associated proteins ZO-1,occludin and claudin-5 in GECsand changes the distribution of tight junction-associated proteins ZO-1,occludin,claudin-5 on GECs,causing them to change from a relatively continuous distribution to a discontinuous distribution at the edge of the cell membrane;silencing mi R-153 significantly increased TEER values,reduced the exudation rate of HRP and significantly enhanced the protein expression of tight junction-associated proteins ZO-1,occludin and claudin-5.6.mi R-377 is underexpressed in GECs.Overexpression of mi R-377 can significantly reduce TEER value,increase HRP exudation rate,and significantly inhibit the protein expression of tight junction-associated proteins ZO-1,occludin and claudin-5 in GECs and changes the distribution of tight junction-associated proteins ZO-1,occludin,claudin-5 on GECs,causing them to change from a relatively continuous distribution to a discontinuous distribution at the edge of the cell membrane;silencing mi R-377 significantly increased TEER values,reduced the exudation rate of HRP and significantly enhanced the protein expression of tight junction-associated proteins ZO-1,occludin and claudin-5.7.Knockdown of MIR17 HG expression in GECs significantly increased the expression of mi R-153 and mi R-377.At the same time,MIR17 HG binds to mi R-153 and mi R-377,and is enriched in the Ago2 complex with mi R-153 and mi R-377,respectively.8.Overexpression of mi R-153 and mi R-377 in GECs with silencing MIR17 HG significantly increased the inhibition of TEER and the promotion of HRP after silencing MIR17 HG.At the same time,it significantly increased the underexpression of tight junction-associated proteins ZO-1,occludin and claudin-5 in GECs after silencing MIR17 HG..In contrast,silencing of mi R-153 and mi R-377 in GECs with silencing MIR17 HG,respectively,can reverse the previous effects.9.FOXR2 is highly expressed in GECs.Overexpression of FOXR2 can significantly increase TEER value and decrease HRP exudation rate.Silencing FOXR2 expression can significantly decrease TEER value and increase HRP exudation.Simultaneous silencing of FOXR2 can significantly inhibit the expression of tight junction-associated protein ZO-1,occludin and claudin-5,and change the distribution of tight junction-associated proteins ZO-1,occludin and claudin-5on GECs from a relatively continuous distribution state to a discontinuous distribution state at the edge of the cell membrane;Overexpression of FOXR2 enhanced the expression of tight junction-associated proteins ZO-1,occludin and claudin-5.11.FOXR2 targets the promoter regions of ZO-1,occludin and caludin-5.12.mi R-153 and mi R-377 targeted FOXR2,respectively.Moreover,overexpression of mi R-153 and mi R-377 in GECs significantly inhibited FOXR2 protein expression.13.Simultaneous double overexpression of FOXR2 and mi R-153 can significantly alter the effect of overexpression of mi R-153 in GECs on reducing TEER and increasing HRP exudation rate,while significantly reversing the inhibitory effect of overexpression of mi R-153 on the expression of tight junction-associated proteins ZO-1,occludin and claudin-5.14.Simultaneous double overexpression of FOXR2 and mi R-377 can significantly alter the effect of overexpression of mi R-377 in GECs on reducing TEER and increasing HRP exudation rate,while significantly reversing the inhibitory effect of overexpression of mi R-377 on the expression of tight junction-associated proteins ZO-1,occludin and claudin-5.15.The in vitro BTB model of overexpressing pi R-DQ590027,mi R-153,mi R-377 in silencing MIR17 HG GECs and the in vitro BTB model of double overexpressing mi R-153 and mi R-377,respectively,can increase the promoting effecr of doxorubicin on the apoptotic of tumor cells.Conclusion: 1.pi R-DQ590027 regulates BTB permeability by binding to MIR17 HG,MIR17HG regulates BTB permeability by binding to mi R-153 and mi R-377.2.mi R-153 and mi R-377 collectively regulate BTB permeability by targeting the 3’-UTR of FOXR2,respectively.3.FOXR2 promotes the transcription of tight junction-associated proteins ZO-1,occludin and claudin-5,up-regulates its expression,and regulates BTB permeability.4.The pi R-DQ590027/MIR17HG/ mi R-137,mi R-377/FOXR2 signal axes play an important role in regulating BTB permeability. |
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