Molecular Mechanism Of KHDRBS3 In Regulating Blood-tumor Barrier Permeability Through CDENND4C/miR-577 Axis

Objective: Gliomas are the most common malignant tumors in the central nervous system with high invasiveness,poor prognosis,and high mortality.At present,the main treatment method is surgery combined with postoperative radiotherapy and chemotherapy.The existence of the blood-tumor barrier(BTB)largely restricts the entry of macromolecular anti-tumor drugs into brain tumor tissues,which seriously affects the effects of drug treatment.Therefore,how to selectively open BTB is very important to improve the chemotherapy effects of glioma.RNA-binding protein(RBP)can bind to non-coding RNAs and regulate their expression and function.The RNA-binding protein KHDRBS3(a signal transduction related factor 3 containing KH RNA-binding domain)regulates the alternative splicing of target genes by binding to KH RNA domains and RNAs.It has been reported that Quaking(QKI),a member of the STAR family and KHDRBS3,not only participates in the formation and reconstruction of embryonic blood vessels,but also regulates the expression of VE-cadherin and ?-catenin at the post-transcription level,thereby affecting the endothelial barrier function.Whether KHDRBS3 could regulate the function of BTB has not been reported.Circular RNA(circRNA)is a non-coding single-stranded RNA molecule without a5' cap and a 3' poly(A)tail structure.CircRNA has a wide range of biological functions,such as interacting with RBPs and regulating the splicing and stability of mRNA,molecular sponges of miRNAs,etc.The circRNA DENND4C(cDENND4C,also known as hsa?circ?0005684)is derived from exons 3-10 of the DENND4 C gene.Studies have shown that cDENND4 C is highly expressed in breast cancer,and the expressions of cDENND4 C in endothelial cells were significantly increased after 12 and 24 h of hypoxia.There has been no report of cDENND4 C on BTB permeability.MicroRNA(miRNA)is a kind of non-coding RNA that usually bind to the 3'untranslated region(UTR)of mRNA and regulate the expressions of target genes at the post-transcriptional level.MiR-577 is down-regulated in glioblastoma,hepatocellular carcinoma,and colorectal cancer.In addition,miRNAs are also involved in regulatingvascular function.For example,miR-181-5p and miR-330-3p are involved in regulating the BTB permeability.It has not yet been confirmed whether miR-577 has a regulatory effect on the permeability of BTB.In this study,the expression levels of KHDRBS3,cDENND4 C,and miR-577 in human glioma microvascular endothelial cells were first detected.Based on this result,the regulatory relationship among the three and the regulatory effects on BTB permeability were studied.By exploring the new targets for selective opening of BTB,it could provide new strategies for chemotherapy of gliomas.Methods: The BTB and blood-brain barrier models were established in vitro,and qRT-PCR was used to detect the expression levels of KHDRBS3,cDENND4 C,and miR-577 in brain microvascular endothelial cells cocultured with astrocytes(AECs)and brain microvascular endothelial cells co-cultured with glioma cells(GECs).After transfection of KHDRBS3 silencing vector,cDENND4 C silencing or overexpression vectors and agomir-577 or antagomir-577 in GECs,qRT-PCR or Western blot was used to detect the transfection efficiencies.Trans-endothelial resistance(TEER)and horseradish peroxidase(HRP)penetration measurements were performed to assess BTB permeability.Western blot and immunofluorescent staining were used to detect changes in the expressions and distributions of tight-junction related proteins ZO-1,occludin and claudin-1.RNA immunoprecipitation and RNA pull-down experiments confirmed the binding between KHDRBS3 and cDENND4 C.The dual luciferase reporter gene system was used to analyze the binding of miR-577 to cDENND4 C,miR-577 to 3'UTRs of ZO-1,occludin,and claudin-1 mRNA,and to identify the binding sites.Flow cytometry was used to analyze the effects of KHDRBS3,cDENND4 C and miR-577 expression regulated alone or in combination on doxorubicin(DOX)induced apoptosis of U87 glioma cells.Results: Compared with the AECs group,KHDRBS3 was highly expressed in GECs.Silencing the expression of KHDRBS3 disrupted the integrity of BTB,increased the BTB permeability,significantly reduced the expressions of tight junction-associated proteins ZO-1,occludin,and claudin-1,and decreased the linear distributions of tight junctions between endothelial cells.cDENND4 C is highly expressed in GECs,and lin-DENND4 C shows no difference in the expressions between AECs and GECs.Aftersilenced the expression of cDENND4 C,the integrity of BTB was destroyed,the BTB permeability was increased,the expressions of ZO-1,occludin and claudin-1 were significantly down-regulated,and the linear distributions of tight junctions between endothelial cells were disrupted.There is the binding between KHDRBS3 and cDENND4 C.Silencing the expression of KHDRBS3 in GECs significantly down-regulated the expression level of cDENND4 C,shortened the half-life of cDENND4 C,and had no effect on the expression of cDENND4 C in newborns.Compared with the KHDRBS3-silenced group,simultaneously silenced the expressions of KHDRBS3 and cDENND4 C in GECs significantly increased the BTB permeability and down-regulated the expressions of tight-junction related proteins.Overexpression of cDENND4 C in KHDRBS3 silenced GECs significantly reduced the BTB permeability,increased the expression levels of tight junction-associated proteins.MiR-577 is lowly expressed in GECs and binds to the 3'UTRs of ZO-1,occludin,and claudin-1 mRNA.Overexpression of miR-577 in GECs significantly increased the permeability of BTB,down-regulated the expressions of tight junction-associated proteins ZO-1,occludin,and claudin-1 and linear distributions between endothelial cells.MiR-577 is capable of targeted binding to cDENND4 C.Compared with the cDENND4 C silencing group,knocking down miR-577 in cDENND4C-silenced GECs significantly reduced the BTB permeability and increased the expression of tight junction-associated proteins.Compared with the DOX group,KHDRBS3 silencing,cDENND4 C silencing or miR-577 overexpression combined with DOX treatments can significantly increase the apoptosis rate of U87 glioma cells.Compared with KHDRBS3 or cDENND4 C silencing combined with DOX group,KHDRBS3 and cDENND4 C double silencing combined with DOX treatment significantly increased the apoptosis rate of U87 glioma cells.Compared with cDENND4 C silencing or miR-577 overexpression combined with DOX group,cDENND4 C silencing and miR-577 overexpression combined with DOX treatment significantly increased the U87 glioma cell apoptosis rate.Conclusion: 1.KHDRBS3 and cDENND4 C are highly expressed in GECs.Silencing KHDRBS3 or cDENND4 C destroyed the integrity of BTB and increased its permeability.2.KHDRBS3 regulated the permeability of BTB via binding to cDENND4 C and increasing its stability.3.MiR-577 is low-expressed in GECs.Overexpression ofmiR-577 negatively regulated the expressions of its target genes,tight junction-associated proteins ZO-1,occludin,and claudin-1,increased the BTB permeability.4.cDENND4 C bond to miR-577 and inhibited its negative regulation of tight junction-associated proteins,affecting the permeability of BTB.5.The regulation of KHDRBS3,cDENND4 C and miR-577 expression alone or in combination promoted DOX across BTB to induce glioma cells apoptosis.