Heng Cai

Endophilin-1 regulates blood-brain barrier permeability by controlling ZO-1 and occludin expression via the EGFR-ERK1/2 pathway.

The blood-brain barrier (BBB) plays a pivotal role in maintenance and regulation of the neural microenvironment. Brain endothelial cells (BECs), held together by tight junctions (TJs), have a primary role in restricting the permeability of the BBB. Endophilin-1 is a multifunctional protein that influences epithelial growth factor receptor (EGFR) endocytosis and degradation and plays an important role in regulating the glomerular filtration barrier in the kidney. Endophilin-1 likely plays a similar role in controlling BBB permeability. In this study, we therefore analyzed the expression and function of endophilin-1 in the human BEC line hCMEC/D3. Our results show that endophilin-1 over-expression reduced the expression of the TJ-associated proteins ZO-1 and occludin and increased the paracellular permeability of hCMEC/D3 cells, whereas silencing of endogenous endophilin-1 yielded the opposite results. Over-expression of ZO-1 and occludin prevented the increase in permeability induced by endophilin-1 over-expression, whereas down-regulation of ZO-1 and occludin prevented the reduction in permeability induced by endophilin-1 silencing. Co-localization and co-immunoprecipitation experiments suggested that endophilin-1 interacts with the EGFR. The levels of EGFR and its downstream effector phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) are significantly decreased when endophilin-1 is over-expressed. Conversely, endophilin-1 down-regulation led to markedly increased levels of these proteins. In addition, the reduced permeability induced by endophilin-1 down-regulation was blocked by AG1478 and PD98059, inhibitors of EGFR and ERK1/2, respectively. Up-regulation of ZO-1 and occludin was blocked by the EGFR and ERK1/2 inhibitors. These results suggest that endophilin-1 regulates BBB permeability by controlling ZO-1 and occludin expression via the EGFR-ERK1/2 pathway in BECs.

Roundabout 4 Regulates Blood–Tumor Barrier Permeability Through the Modulation of ZO-1, Occludin, and Claudin-5 Expression

Abstract The blood–tumor barrier (BTB) restricts the delivery of chemotherapeutic drug molecules to tumor tissues. We found that the endothelial cell (EC) receptor molecule Roundabout 4 (Robo4) is endogenously expressed in human brain microvascular ECs and that it is upregulated in a BTB model of glioma cocultured ECs. Knockdown of Robo4 in this BTB model increased permeability; short hairpin RNA targeting Robo4 (shRobo4) led to decreased transendothelial electric resistance values, increased BTB permeability, and downregulated expression of the EC tight junction proteins ZO-1, occludin, and claudin-5. Roundabout 4 influenced BTB permeability via binding with its ligand, Slit2. Short hairpin RNA targeting Robo4 also increased matrix metalloproteinase-9 (MMP-9) activity and expression in glioma cocultured ECs; pretreatment with the MMP inhibitor GM6001 partially blocked the effects of shRobo4 on the transendothelial electric resistance values and ZO-1 and occludin expression. Short hairpin RNA targeting Robo4 also upregulated the phosphorylation of Src and Erk1/2; the Src inhibitor PP2 and the Erk1/2 inhibitor PD98059 blocked shRobo4-mediated alteration in ZO-1 and occludin expression. Together, our results indicate that knockdown of Robo4 increased BTB permeability by reducing EC tight junction protein expression, and that the Src–Erk1/2–MMP-9 signal pathways are involved in this process. Thus, Robo4 may represent a useful future therapeutic target for enhancing BTB permeability.

Knockdown of long non-coding RNA XIST increases blood–tumor barrier permeability and inhibits glioma angiogenesis by targeting miR-137

Antiangiogenic therapy plays a significant role in combined glioma treatment. However, poor permeability of the blood–tumor barrier (BTB) limits the transport of chemotherapeutic agents, including antiangiogenic drugs, into tumor tissues. Long non-coding RNAs (lncRNAs) have been implicated in various diseases, especially malignant tumors. The present study found that lncRNA X-inactive-specific transcript (XIST) was upregulated in endothelial cells that were obtained in a BTB model in vitro. XIST knockdown increased BTB permeability and inhibited glioma angiogenesis. The analysis of the mechanism of action revealed that the reduction of XIST inhibited the expression of the transcription factor forkhead box C1 (FOXC1) and zonula occludens 2 (ZO-2) by upregulating miR-137. FOXC1 decreased BTB permeability by increasing the promoter activity and expression of ZO-1 and occludin, and promoted glioma angiogenesis by increasing the promoter activity and expression of chemokine (C–X–C motif) receptor 7b (CXCR7). Overall, the present study demonstrates that XIST plays a pivotal role in BTB permeability and glioma angiogenesis, and the inhibition of XIST may be a potential target for the clinical management of glioma.

Long non-coding RNA NEAT1 regulates permeability of the blood-tumor barrier via miR-181d-5p-mediated expression changes in ZO-1, occludin, and claudin-5

The blood-tumor barrier (BTB) constitutes an efficient organization of tight junctions that limits the delivery of chemotherapeutic drugs to brain tumor tissues and impacts the treatment of glioma. Long non-coding RNAs (lncRNAs) are non-protein coding RNAs regulating gene expression, some lncRNAs play a crucial role in BTB permeability. However, the function of lncRNAs in BTB permeability is still largely unclear. Here, we have identified lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1), was remarkably up-regulated in glioma endothelial cells (GECs) obtained from an in vitro BTB model. Knockdown of NEAT1 impaired the integrity and increased the permeability of the BTB, accompanied by downregulation of expression of the tight junction proteins ZO-1, occludin and claudin-5 in GECs. Both bioinformatics data and results of luciferase reporter assays demonstrated that NEAT1 influenced BTB permeability by binding to miR-181d-5p. Knockdown of NEAT1 also down-regulated the expression of sex determining region Y-box protein 5 (SOX5), which was defined as a direct and functional downstream target of miR-181d-5p. SOX5 interacts with the promoter region of ZO-1, occludin and claudin-5 in GECs. In conclusion, knockdown of NEAT1 increased BTB permeability by binding to miR-181d-5p and then reducing tight junction protein expression by targeting SOX5. These results suggest an important role for NEAT1 in regulating BTB permeability and provide an additional strategy for treating glioma.

Role of HCP5-miR-139-RUNX1 Feedback Loop in Regulating Malignant Behavior of Glioma Cells.

Aberrant expression of long noncoding RNAs has recently been reported in tumorigenesis and plays a pivotal role in regulating malignant behavior of cancers. In this study, we confirmed that the long noncoding RNAs human histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) was up-regulated in glioma tissues as well as in U87 and U251 cells. Knockdown of HCP5 inhibited the malignant biological behavior of glioma cells by reducing proliferation, migration and invasion, and inducing apoptosis. HCP5 regulated the malignant behavior of glioma cells by binding to microRNA-139, which functions as a tumor suppressor. Moreover, knockdown of HCP5 down-regulated Runt-related transcription factor 1, a direct and functional downstream target of microRNA-139 that is involved in microRNA-139-mediated tumor-suppressive effects in glioma cells. Runt-related transcription factor 1 increased promoter activities and upregulated expression of the oncogenic gene astrocyte elevated gene-1 (AEG-1). Runt-related transcription factor 1 also increased the promoter activities and expression of HCP5, which showed a positive feedback loop in regulating the malignant behavior of glioma cells. In conclusion, this study demonstrated that the HCP5-microRNA-139- Runt-related transcription factor 1 feedback loop plays a pivotal role in regulating the malignant behavior of glioma cells, which may provide a potential therapeutic strategy for treating glioma.

MicroRNA-107 prevents amyloid-beta induced blood-brain barrier disruption and endothelial cell dysfunction by targeting Endophilin-1

The disruption of blood-brain barrier (BBB) and endothelial cell dysfunction, associated with the cerebrovascular deposition of the amyloid-beta (Abeta) protein, have been characterized as the key pathological characteristics in Alzheimers disease (AD). In various biologic processes of AD, researchers have proven that mircroRNAs (miRNAs) play critical roles. However, the role and function of miRNAs in the disruption of BBB of AD still remain unclear. Here, we found that mircroRNA-107 (miR-107) is endogenously expressed in human brain microvascular endothelial cells (ECs) of BBB model, while it is significantly down-regulated in ECs pre-incubated with Abeta. Abeta significantly impairs the integrity, increases the permeability of BBB, inhibits the viability of endothelial cells (ECs), and meanwhile down-regulates the expression of tight junction proteins ZO-1, Occludin and Claudin-5. Overexpression of miR-107 largely abrogated Abeta-induced disruption of BBB and endothelial cell dysfunction. Furthermore, overexpression of miR-107 also down-regulates endophilin-1, which is involved in the regulation of BBB permeability and the expression of ZO-1, Occludin, and Claudin-5. Both bioinformatics and luciferase reporter assays demonstrated that Endophilin-1 was a direct and functional downstream target of miR-107. In conclusion, our results indicate that overexpression of miR-107 is able to prevent Abeta-induced blood-brain barrier disruption and endothelial cell dysfunction by targeting endophilin-1.

MiR-383 inhibits proliferation, migration and angiogenesis of glioma-exposed endothelial cells in vitro via VEGF-mediated FAK and Src signaling pathways

Malignant glioma is undoubtedly the most vascularized tumor of central nervous system. Angiogenesis, playing a predominant role in tumor progression, is widely considered as a key point of tumor treatment. The aim of this study was to investigate the potential effects of miR-383 on proliferation, migration, tube formation and angiogenesis of glioma-exposed endothelial cells (GECs) in vitro and to further elucidate its possible molecular mechanisms. The expression of miR-383 in GECs was significantly downregulated compared with that in normal endothelial cells (ECs). Overexpression of miR-383 dramatically inhibited the proliferation, migration, tube formation and spheroid-based angiogenesis of GECs in vitro. Dual-luciferase reporter results demonstrated vascular endothelial growth factor (VEGF) is a target gene of miR-383. Furthermore, overexpression or silencing of either miR-383 or VEGF was performed simultaneously to further clarify that miR-383 inhibited proliferation, migration and angiogenesis of GECs in vitro by targeting VEGF. Finally, VEGF/VEGFR2-mediated FAK and Src signaling pathways might contribute to anti-angiogenesis of GECs. In conclusion, our present study indicated that miR-383 inhibits proliferation, migration and angiogenesis of GECs in vitro via VEGF/VEGFR2-mediated FAK and Src signaling pathways, which would draw growing attention to miR-383c as a potential therapeutical target of glioma.

Knockdown of SOX2OT inhibits the malignant biological behaviors of glioblastoma stem cells via up-regulating the expression of miR-194-5p and miR-122

BackgroundAccumulating evidence has highlighted the potential role of long non-coding RNAs (lncRNAs) in the biological behaviors of glioblastoma stem cells (GSCs). Here, we elucidated the function and possible molecular mechanisms of the effect of lncRNA-SOX2OT on the biological behaviors of GSCs.ResultsReal-time PCR demonstrated that SOX2OT expression was up-regulated in glioma tissues and GSCs. Knockdown of SOX2OT inhibited the proliferation, migration and invasion of GSCs, and promoted GSCs apoptosis. MiR-194-5p and miR-122 were down-regulated in human glioma tissues and GSCs, and miR-194-5p and miR-122 respectively exerted tumor-suppressive functions by inhibiting the proliferation, migration and invasion of GSCs, while promoting GSCs apoptosis. Knockdown of SOX2OT significantly increased the expression of miR-194-5p and miR-122 in GSCs. Dual-luciferase reporter assay revealed that SOX2OT bound to both miR-194-5p and miR-122. SOX3 and TDGF-1 were up-regulated in human glioma tissues and GSCs. Knockdown of SOX3 inhibited the proliferation, migration and invasion of GSCs, promoted GSCs apoptosis, and decreased TDGF-1 mRNA and protein expression through direct binding to the TDGF-1 promoter. Over-expression of miR-194-5p and miR-122 decreased the mRNA and protein expression of SOX3 by targeting its 3’UTR. Knockdown of TDGF-1 inhibited the proliferation, migration and invasion of GSCs, promoted GSCs apoptosis, and inhibited the JAK/STAT signaling pathway. Furthermore, SOX3 knockdown also inhibited the SOX2OT expression through direct binding to the SOX2OT promoter and formed a positive feedback loop.ConclusionThis study is the first to demonstrate that the SOX2OT-miR-194-5p/miR-122-SOX3-TDGF-1 pathway forms a positive feedback loop and regulates the biological behaviors of GSCs, and these findings might provide a novel strategy for glioma treatment.

circ-SHKBP1 Regulates the Angiogenesis of U87 Glioma-Exposed Endothelial Cells through miR-544a/FOXP1 and miR-379/FOXP2 Pathways

Circular RNAs (circRNAs) are a type of endogenous non-coding RNAs, which have been considered to mediate diverse tumorigenesis including angiogenesis. The present study aims to elucidate the potential role and molecular mechanism of circ-SHKBP1 in regulating the angiogenesis of U87 glioma-exposed endothelial cells (GECs). The expression of circ-SHKBP1, but not linear SHKBP1, was significantly upregulated in GECs compared with astrocyte-exposed endothelial cells (AECs). circ-SHKBP1 knockdown inhibited the viability, migration, and tube formation of GECs dramatically. The expressions of miR-379/miR-544a were downregulated in GECs, and circ-SHKBP1 functionally targeted miR-544a/miR-379 in an RNA-induced silencing complex (RISC) manner. Dual-luciferase reporter assay demonstrated that forkhead box P1/P2 (FOXP1/FOXP2) were targets of miR-544a/miR-379. The expressions of FOXP1/FOXP2 were upregulated in GECs, and silencing of FOXP1/FOXP2 inhibited the viability, migration, and tube formation of GECs. Meanwhile, FOXP1/FOXP2 promoted angiogenic factor with G patch and FHA domains 1 (AGGF1) expression at the transcriptional level. Furthermore, knockdown of AGGF1 suppressed the viability, migration, and tube formation of GECs via phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK)1/2 pathways. Taken together, the present study demonstrated that circ-SHKBP1 regulated the angiogenesis of GECs through miR-544a/FOXP1 and miR-379/FOXP2 pathways, and these findings might provide a potential target and effective strategy for combined therapy of gliomas.

Long Non-coding RNA LINC00339 Stimulates Glioma Vasculogenic Mimicry Formation by Regulating the miR-539-5p/TWIST1/MMPs Axis

Glioma is recognized as a highly angiogenic malignant brain tumor. Vasculogenic mimicry (VM) greatly restricts the therapeutic effect of anti-angiogenic tumor therapy for glioma patients. However, the molecular mechanisms of VM formation in glioma remain unclear. Here, we demonstrated that LINC00339 was upregulated in glioma tissue as well as in glioma cell lines. The expression of LINC00339 in glioma tissues was positively correlated with glioma VM formation. Knockdown of LINC00339 inhibited glioma cell proliferation, migration, invasion, and tube formation, meanwhile downregulating the expression of VM-related molecular MMP-2 and MMP-14. Furthermore, knockdown of LINC00339 significantly increased the expression of miR-539-5p. Both bioinformatics and luciferase reporter assay revealed that LINC00339 regulated the above effects via binding to miR-539-5p. Besides, overexpression of miR-539-5p resulted in decreased expression of TWIST1, a transcription factor known to play an oncogenic role in glioma and identified as a direct target of miR-539-5p. TWIST1 upregulated the promoter activities of MMP-2 and MMP-14. The in vivo study showed that nude mice carrying tumors with knockdown of LINC00339 and overexpression of miR-539-5p exhibited the smallest tumor volume through inhibiting VM formation. In conclusion, LINC00339 may be used as a novel therapeutic target for VM formation in glioma.