| Objective: Gliomas are the most frequent primary malignant tumor in the human brain.The current treatment for gliomas is surgical resection,combined with postoperative radiotherapy and chemotherapy.The blood tumor barrier(BTB)greatly restricts the delivery of chemotherapeutic drugs into tumor tissue and reduces the efficacy of chemotherapy.Therefore,the selective opening of BTB to increase drug concentrations in brain tumor tissues is essential to improve the effectiveness of chemotherapy for malignant gliomas.PIWI(P-element-induced wimpy testis)proteins belong to the Argonaute(AGO)family,which are involved in the progress of cancer.PIWI proteins recognize and bind PIWI interacting RNAs(piRNAs),that constitutes the piRNA-induced silencing complex(piRISC).They paly important role in epigenetic regulation,the silencing of transposable elements et al.PIWIL1(Piwi‐like RNA‐mediated gene silencing 1)is a member of PIWI family,and promote glioma proliferation,migration and inhibit glioma apoptosis.However,the expression and function of PIWIL1 in brain microvascular endothelial cells have not been reported.piRNA(PIWI-interacting RNAs)are non-coding RNAs of 23-34 nucleotides(nt)in length.Emerging studies have shown that piRNAs are crucial in cancer development and progression and may use as a molecular target for the treatment of cancer.However,the function of piR-DQ593109 in cancer remain unclear.RNA binding protein(RBPs)regulate many crucial cellar processes,including RNA splicing,stability,RNA decay,and translation.RBFOX1 is a member of RBFOX family,selectively expressed in brain,heart,and skeletal muscle,and regulate the process of cancer.Besides,deletions of RBFOX1 have been found in patients with neurological disorders,such as epilepsy,and autism.Also,RBFOX1 is downregulated in glioblastoma multiforme.While,the expression and function of RBFOX1 in brain microvascular endothelial cells have not been reported.The present study aims to explore the expression and function of PIWIL1/piR-DQ593109,and RBFOX1 in glioma microvascular endothelial cells.And further investigate the mechanism of PIWIL1/piR-DQ593109,and RBFOX1 regulating BTB permeability.Which may provide novel strategies for the treatment of glioma.Methods: 1.Establish the blood brain barrier(BBB)model in vitro and BTB model in vitro;2.The real-time PCR was performed to detect the expression level of PIWIL1,piR-DQ593109,MEG3,miR-330-5p,RUNX3,RBFOX1,LINC00673,and MAFF;3.We performed the western blot to detect the expression of PIWIL1,RUNX3,MAFF,ZO-1,occludin,and claudin-5;4.Stable transfection of PIWIL1 silent plasmid,MEG3 overexpression plasmid,RBFOX1 overexpression plasmid,RBFOX1 silent plasmid,LINC00673 silent plasmid,LINC00673 overexpression plasmid,MAFF overexpression plasmid,and MAFF silent plasmid in glioma microvascular endothelial cells(GECs),respectively;transiently transfection of antagomirpiR-DQ593109,agomir-330-5p,antagomir-330-5p in GECs;5.Trans-endothelial resistance(TEER)assay was performed to determine the integrity of BTB;6.Horseradish peroxidase(HRP)assay was used to determine the permeability of BTB;7.The expression and distribution of tight junction related proteins ZO-1,occludin,claudin-5 were detected by Immunofluorescence staining assay;8.The binding sites of piR-DQ593109 and MEG3,MEG3 and miR-330-5p,miR-330-5p and RUNX3 3’UTR,RUNX3 and ZO-1,occludin,claudin-5 promoter,as well as LINC00673 and MAFF,MAFF and ZO-1,occludin,claudin-5 promoter were detected and verified by Dual luciferase reporter gene experiments;9.The binding of piR-DQ593109,MEG3 and PIWIL1,MEG3,mi R-330-5p and Ago2,as well as RBFOX1 and LINC00673,LINC00673,MAFF and STAU1 were detected by RNA binding protein immunoprecipitation assay;10.The targeted binding of RUNX3 and ZO-1,occludin,claudin-5 promoter,MAFF and ZO-1,occludin,claudin-5 promoter were verified by Chromatin immunoprecipitation(ChIP)assay;11.The opening and application of BTB was detected by Flow cytometry technology.Results: 1.PIWIL1 and piR-DQ593109 wrer upregulated in GECs,knockdown of PIWIL1,piR-DQ593109,and PIWIL1/piR-DQ593109 complex significantly decreased the TEER value,increased the HRP flux,and decreased the expression of tight junction related proteins ZO-1,occludin,claudin-5.As well as,the distribution of ZO-1,occludin,claudin-5 was changed to discontinues on cell boundaries.2.MEG3 was downregulated in GECs,overexpression of MEG3 significantly increased the BTB permeability,and MEG3 mediated the function of PIWIL1/piR-DQ593109 complex on BTB permeability in a sequence-specific manner.3.MiR-330-5p was targeted MEG3 in GECs,co-transfection of overexpression of MEG3 and overexpression of miR-330-5p,significantly reversed the decreased in TEER value,the increases in HRP flux,as well as the expression decreased in ZO-1,occludin,claudin-5 induced by overexpression of MEG3 alone.4.miR-330-5p was negative regulated the target gene RUNX3’ expression.Co-transfection of overexpression of miR-330-5p and RUNX3,significantly reversed he decreased in TEER value,the increases in HRP flux,as well as the expression decreased in ZO-1,occludin,claudin-5 induced by miR-330-5p overexpression alone in GECs.5.RUNX3 was bound to the promoter of tight junction related proteins ZO-1,occludin,claudin-5,and inhibit the activity of promoter.6.RBFOX1 was downregulated in GECs,RBFOX1 overexpression decreased the TEER value,increased the HRP flux,and decreased the expression of tight junction related proteins ZO-1,occludin,claudin-5.As well as,the distribution of ZO-1,occludin,claudin-5 was changed to discontinues on cell boundaries flowing RBFOX1 overexpression;knockdown of RBFOX1 shows the opposite results.7.LINC00673 was upregulated in GECs,knockdown of LINC00673 significantly decreased the TEER value,increased the HRP flux,and decreased the expression of tight junction related proteins ZO-1,occludin,claudin-5.As well as,the distribution of ZO-1,occludin,claudin-5 was changed to discontinues on cell boundaries flowing knockdown of LINC00673;LINC00673 overexpression shows the opposite results.8.Overexpression of RBFOX1 significantly decreased the stability of LINC00673 in GECs,and LINC00673 was involved in the regulation of BTB permeability by RBFOX1.9.LINC00673 regulated the target gene expression of MAFF through SMD process,and MAFF was involved in the regulation of LINC00673 on BTB permeability.10.MAFF overexpression significantly increased the BTB permeability,while knockdown of MAFF shows the opposite results in GECs.11.MAFF bound to the promoter of tight junction related proteins ZO-1,occludin,claudin-5,and inhibited transcription avtivity.12.Stable transfection of RBFOX1 overexpression,LINC00673 knockdown,and co-transfection of RBFOX1 overexpression and LINC00673 knockdown combined with doxorubicin(Dox),respectively,promoted the apoptotic of glioma cells.Conclusion: 1.PIWIL1 reduces the expression of MEG3 through PIWIL1/ piR-DQ593109 to regulate the BTB permeability.2.MEG3 regulated the BTB permeablity by targeting miR-330-5p to reduce the expression of RUNX3.3.RUNX3 directly binds to the promoter regions of ZO-1,occludin,claudin-5 respectively,transcriptionally inhibits its expression and regulates BTB permeability.4.The PIWIL1/piR-DQ593109/MEG3/miR-330-5p/RUNX3 signal axis play a crucial role in regulating BTB permeability.5.RBFOX1 increases BTB permeability by reducing the the stability of LINC00673.6.LINC00673 attenuates the degradation of MAFF through the SMD pathway,promotes the transcriptional inhibition of the tight junction proteins ZO-1,occludin,claudin-5 by MAFF and increases BTB permeability.7.The RBFOX1/LINC00673/MAFF pathway play an important role in regulation of BTB permeability. |
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