Dissolving Polymer Microneedles for Transdermal Delivery of Insulin.

It's of great significance to develop insulin-loaded dissolving microneedles (MNs) which are fabricated with various methods and materials for transdermal delivery of insulin to effectively and efficiently treat diabetes. In this work, we present a kind of FITC-insulin tip-loaded dissolving MNs fabricated with the mixture of polyvinyl alcohol (PVA) and sucrose using homemade PDMS MNs mold under vacuum conditions. The uniform appearance of MN arrays contributes to controlling the drug dosage well as required. Sufficient mechanical strength for penetrating tough stratum corneum can be obtained by vacuum frozen-drying for at least 6 h after peeling MNs off the mold. About 90% of the FITC-insulin is localized in the conical MN tips and can be released into the skin within 2 min after insertion. The in vivo insulin absorption study and hypoglycemic effect in diabetic mice demonstrate that the proposed insulin-loaded MNs can efficiently deliver the insulin to the systemic circulation and exhibit a similar effect to hypodermic injection on hypoglycemic administration. Together these results suggested that the efficient MN fabrication process proposed in this work shows great potential for mass production and practical application of drug-loaded dissolving MNs in the future.

Engineered bovine serum albumin-based nanoparticles with pH-sensitivity for doxorubicin delivery and controlled release.

In this work, we prepared a stimuli-responsive system for drug delivery and controlled release by engineering the bovine serum albumin (BSA). The doxorubicin (DOX)-loaded BSA nanoparticles (NPs) were conveniently prepared using desolvation method, followed by crosslinking through Schiff base bonds, leading to pH-sensitive DOX-loaded system (DOXs@BSA NPs). The resulted DOXs@BSA NPs showed high drug loading capacity (21.4%), and the particle size was about 130 nm with narrow polydispersity and high negative surface charge (-20.5 mV). The pH-sensitivity of DOXs@BSA NPs was evidenced by the size changes and charge reversal after incubation at different pH values. The DOXs@BSA NPs showed high serum stability which indicated the prolonged circulation time. The in vitro drug release experiment showed that the release of DOX was obviously accelerated by acidity because of disassembly of NPs induced by cleavage of Schiff base bonds. The drug release mechanism was thoroughly studied using a semi-empirical model, further confirming the pH played an important role in drug controlled release process. The results of cytotoxicity assay revealed that DOXs@BSA NPs exhibited much higher toxic effects for tumor cells in comparison to the free DOX control. Collectively, these results demonstrated that DOXs@BSA NPs might be potential application for drug delivery and controlled release in cancer chemotherapy. Moreover, this work also showed that preparation of stimuli-responsive drug delivery system by engineering the commercial biomaterials could be a promising method to develop multi-functional nanomedicine.

RBFOX1 Regulates the Permeability of the Blood-Tumor Barrier via the LINC00673/MAFF Pathway.

The blood-tumor barrier limits the delivery of therapeutic drugs to brain tumor tissues. Selectively opening the blood-tumor barrier is considered crucial for effective chemotherapy of glioma. RNA-binding proteins have emerged as crucial regulators in various biologic processes. This study found that RNA-binding Fox-1 homolog 1 (RBFOX1) was downregulated in glioma vascular endothelial cells derived from glioma tissues, and in glioma endothelial cells obtained by co-culturing endothelial cells with glioma cells. Overexpression of RBFOX1 impaired the integrity of the blood-tumor barrier and increased its permeability. Additionally, RBFOX1 overexpression decreased the expression of tight junction proteins ZO-1, occludin, and claudin-5. Subsequent analysis of the mechanism indicated that the overexpression of RBFOX1 increased musculoaponeurotic fibrosarcoma protein basic leucine zipper [bZIP] transcription factor F (MAFF) expression by downregulating LINC00673, which stabilized MAFF messenger RNA (mRNA) through Staufen1-mediated mRNA decay. Moreover, MAFF could bind to the promoter region and inhibit the promoter activities of ZO-1, occludin, and claudin-5, which reduced its expression. The combination of RBFOX1 upregulation and LINC00673 downregulation promoted doxorubicin delivery across the blood-tumor barrier, resulting in apoptosis of glioma cells. In conclusion, this study indicated that overexpression of RBFOX1 increased blood-tumor barrier permeability through the LINC00673/MAFF pathway, which might provide a new useful target for future enhancement of blood-tumor barrier permeability.

Sandwich-Like C@SnS@TiO2 Anodes with High Power and Long Cycle for Li-Ion Storage.

Up to now, high energy density batteries can be easily achieved by using alloys or conversion materials with high theoretical capacities (such as silicon-based and tin-based materials). However, these anode materials tend to sacrifice power densities while maintaining high energy densities. Herein, a sandwich-like C@SnS@TiO2 anode with both high capacity and high power is designed by controlling a close integration between interfacial layers. The volume expansion of the middle layer of the SnS in the C@SnS@TiO2 anode is greatly constrained by a synergetic interaction of the TiO2 core and the carbon shell. From the results of the real-time dynamic evolution of electrode thickness during charging and discharging processes, the sandwich-like C@0.5SnS@TiO2 has a max expansion rate of 11.5% in the first lithiation, which is much lower than that of pristine SnS (89.2%), and the expansion of C@0.5SnS@TiO2 is basically reversible in the following charging/discharging processes. As a result, the sandwich-like C@0.5SnS@TiO2 anode delivers a stable capacity of 660mAh g-1 at 50 mA g-1 and manifests an excellent rate capability, with a capacity of 357.2 mAh g-1 at 5A g-1 and a recovery ability of nearly 100%. In addition, it exhibits an outstanding long lifespan, retaining 95.6% capacity after 2500 cycles at 1A g-1. This work presents a durable tin-based anode with moderate capacity for high-energy batteries and offers some ideas for the delicate study of materials with severe expansion during circulation.

Biosynthetic CircRNA_001160 induced by PTBP1 regulates the permeability of BTB via the CircRNA_001160/miR-195-5p/ETV1 axis.

The presence of the blood-tumor barrier (BTB) severely impedes the transport of anti-neoplasm drugs to the central nervous system, affecting the therapeutic effects of glioma. Glioma endothelial cells (GECs) are the main structural basis of the BTB. Circular RNA is considered to be an important regulator of endothelial cell growth. In this study, we found that polypyrimidine tract binding protein 1 (PTBP1) and circRNA_001160 were remarkably upregulated in GECs. Knockdown of PTBP1 or circRNA_001160 significantly increased BTB permeability, respectively. As a molecular sponge of miR-195-5p, circRNA_001160 attenuated its negative regulation of the target gene ETV1 by adsorbing miR-195-5p. In addition, ETV1 was overexpression in GECs. ETV1 bounded to the promoter regions of tight junction-related proteins and increased the promoter activities, which significantly promoted the expression levels of tight junction-related proteins. The present study showed that the combined application of PTBP1, circRNA_001160, and miR-195-5p with the anti-tumor drug Dox effectively promoted Dox through BTB and extremely induced the apoptosis of glioma cells. Our results demonstrated that the PTBP1/circRNA_001160/miR-195-5p/ETV1 axis was critical in the regulation of BTB permeability and provided new targets for the treatment of glioma.

Natural silibinin modulates amyloid precursor protein processing and amyloid-ß protein clearance in APP/PS1 mice.

Silibinin has been shown to attenuate cognitive dysfunction and inhibit amyloid-beta (Aß) aggregation in Alzheimer's disease (AD) models. However, the underlying mechanism by which silibinin improves cognition remains poorly understood. In this study, we investigated the effect of silibinin on ß-secretase levels, Aß enzymatic degradation, and oxidative stress in the brains of APP/PS1 mice with cognitive impairments. Oral administration of silibinin for 2 months significantly attenuated the cognitive deficits of APP/PS1 mice in the Y-maze test, novel object recognition test, and Morris water maze test. Biochemical analyses revealed that silibinin decreased Aß deposition and the levels of soluble Aß1-40/1-42 in the hippocampus by downregulating APP and BACE1 and upregulating NEP in APP/PS1 mice. In addition, silibinin decreased the MDA content and increased the activities of the antioxidant enzymes CAT, SOD, and NO. Based on our findings, silibinin is a potentially promising agent for preventing AD-associated Aß pathology.

MOV10 binding circ-DICER1 regulates the angiogenesis of glioma via miR-103a-3p/miR-382-5p mediated ZIC4 expression change.

BACKGROUND: RNA binding proteins (RBPs) have been reported to interact with RNAs to regulate gene expression. Circular RNAs (circRNAs) are a type of endogenous non-coding RNAs, which involved in the angiogenesis of tumor. The purpose of this study is to elucidate the potential roles and molecular mechanisms of MOV10 and circ-DICER1 in regulating the angiogenesis of glioma-exposed endothelial cells (GECs). METHODS: The expressions of circ-DICER1, miR-103a-3p and miR-382-5p were detected by real-time PCR. The expressions of MOV10, ZIC4, Hsp90 and PI3K/Akt were detected by real-time PCR or western blot. The binding ability of circ-SHKBP1 and miR-544a / miR-379, ZIC4 and miR-544a / miR-379 were analyzed with Dual-Luciferase Reporter System or RIP experiment. The direct effects of ZIC4 on the Hsp90ß promoter were analyzed by the ChIP experiment. The cell viability, migration and tube formation in vitro were detected by CCK-8, Transwell assay and Matrigel tube formation assay. The angiogenesis in vivo was evaluated by Matrigel plug assay. Student's t-test (two tailed) was used for comparisons between two groups. One-way analysis of variance (ANOVA) was used for multi-group comparisons followed by Bonferroni post-hoc analysis. RESULTS: The expressions of RNA binding proteins MOV10, circ-DICER1, ZIC4, and Hsp90ß were up-regulated in GECs, while miR103a-3p/miR-382-5p were down-regulated. MOV10 binding circ-DICER1 regulated the cell viability, migration, and tube formation of GECs. And the effects of both MOV10 and circ-DICER1 silencing were better than the effects of MOV10 or circ-DICER1 alone silencing. In addition, circ-DICER1 acts as a molecular sponge to adsorb miR-103a-3p / miR-382-5p and impair the negative regulation of miR-103a-3p / miR-382-5p on ZIC4 in GECs. Furthermore, ZIC4 up-regulates the expression of its downstream target Hsp90ß, and Hsp90 promotes the cell viability, migration, and tube formation of GECs by activating PI3K/Akt signaling pathway. CONCLUSIONS: MOV10 / circ-DICER1 / miR-103a-3p (miR-382-5p) / ZIC4 pathway plays a vital role in regulating the angiogenesis of glioma. Our findings not only provides novel mechanisms for the angiogenesis of glioma, but also provide potential targets for anti-angiogenesis therapies of glioma.

Mechanism of piR-DQ590027/MIR17HG regulating the permeability of glioma conditioned normal BBB.

BACKGROUND: The blood-brain barrier (BBB) strongly restricts the entry of anti-glioma drugs into tumor tissues and thus decreases chemotherapy efficacy. Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumor form. Because regulation of the effect of noncoding RNA on BBB function is attracting growing attention, we investigated the effects of noncoding RNA on the permeability of glioma conditioned normal BBB and the mechanism involved using PIWI-associated RNA piR-DQ590027 as a starting point. METHODS: The mRNA levels of MIR17HG, miR-153, miR-377, ZO-1, occludin, and claudin-5 were determined using real-time PCR. Transient cell transfection was performed using Lipofectamine 3000 reagent. TEER and HRP flux were applied to measure the permeability of glioma conditioned normal BBB. Western blotting and immunofluorescence assays were used to measure ZO-1, occludin, and claudin-5 levels. Reporter vector construction and a luciferase reporter assay were performed to detect the binding sites of MIR17HG and piR-DQ590027, MIR17HG and miR-153 (miR-377), and FOXR2 and miR-153 (miR-377). RNA immunoprecipitation was used to test the interaction between miR-153 (miR-377) and its target proteins. Chromatin immunoprecipitation was performed to detect the interaction between the transcription factor FOXR2 and ZO-1, occludin, and claudin-5. RESULTS: piR-DQ590027 was expressed at low levels in glioma-conditioned ECs (GECs) of the in vitro glioma conditioned normal BBB model. Overexpression of piR-DQ590027 down-regulated the expressions of ZO-1, occludin, and claudin-5 and increased the permeability of glioma conditioned normal BBB. MIR17HG had high expression in GECs but miR-153 and miR-377 had low expression. piR-DQ590027 bound to and negatively regulated MIR17HG. FOXR2 was a downstream target of miR-153 and miR-377; MIR17HG bound separately to miR-153 and miR-377 and negatively regulated their ability to mediate FOXR2 expression. FOXR2 associated with the promoter regions of ZO-1, occludin, and claudin-5 in GECs to promote their transcription. CONCLUSION: The piR-DQ590027/MIR17HG/miR-153 (miR-377)/FOXR2 pathway plays an important role in regulating glioma conditioned normal BBB permeability and provides a new target for the comprehensive treatment of glioma.

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.

PIWIL1/piRNA-DQ593109 Regulates the Permeability of the Blood-Tumor Barrier via the MEG3/miR-330-5p/RUNX3 Axis.

The blood-tumor barrier (BTB) restricts the efficient delivery of anti-glioma drugs to cranial glioma tissues. Increased BTB permeability may allow greater delivery of the therapeutic agents. Increasing evidence has revealed that PIWI proteins and PIWI-interacting RNAs (piRNAs) play an important role in tumor progression. However, whether PIWI proteins and piRNAs regulate BTB permeability remains unclear. In the present study, we demonstrated that the PIWIL1/piRNA-DQ593109 (piR-DQ593109) complex was the predominant regulator of BTB permeability. Briefly, PIWIL1 was upregulated in glioma endothelial cells (GECs). Furthermore, piR-DQ593109 was also overexpressed in GECs, as revealed via a piRNA microarray. Downregulation of PIWIL1 or piR-DQ593109 increased the permeability of the BTB. Moreover, PIWIL1 and piR-DQ593109, which formed a piRNA-induced silencing complex, degraded the long non-coding RNA maternally expressed 3 (MEG3) in a sequenced-dependent manner. Furthermore, restoring MEG3 released post-transcriptional inhibition of Runt related transcription factor 3 (RUNX3) by sponging miR-330-5p. In addition, RUNX3 bounded to the promoter regions and reduced the promoter activities of ZO-1, occludin, and claudin-5, which significantly impaired the expression levels of ZO-1, occludin, and claudin-5. In conclusion, downregulating PIWIL1 and piR-DQ593109 increased BTB permeability through the MEG3/miR-330-5p/RUNX3 axis. These data may provide insight into glioma treatment.

Long Non-Coding RNA HOXA-AS2 Regulates Malignant Glioma Behaviors and Vasculogenic Mimicry Formation via the MiR-373/EGFR Axis.

BACKGROUND/AIMS: Vasculogenic mimicry (VM) has been reported to be a novel glioma neovascularization process. Anti-VM therapy provides new insight into glioma clinical management. In this study, we revealed the role of the long non-coding RNA HOXA cluster antisense RNA 2 (HOXA-AS2) in malignant glioma behaviors and VM formation. METHODS: Quantitative real-time PCR was performed to determine the expression levels of HOXA-AS2 in glioma samples and glioblastoma cell lines. CD34-periodic acid-Schiff dual-staining was performed to assess VM in glioma samples. CCK-8, transwell, and Matrigel tube formation assays were performed to measure the effects of HOXA-AS2 knockdown on cell viability, migration, invasion, and VM tube formation, respectively. RNA immunoprecipitation, dual-luciferase reporter and Western blot assays were performed to explore the molecular mechanisms underlying the functions of HOXS-AS2 in glioblastoma cells. A nude mouse xenograft model was used to investigate the role of HOXA-AS2 in xenograft glioma growth and VM density. Student's t-tests, one-way ANOVAs followed by Bonferroni posthoc tests, and chi-square tests were used for the statistical analyses. RESULTS: HOXA-AS2 was upregulated in glioma samples and cell lines and was positively correlated with VM. HOXA-AS2 knockdown attenuated cell viability, migration, invasion, and VM formation in glioma cells and inhibited the expression of vascular endothelial-cadherin (VE-cadherin), as well as the expression and activity of matrix metalloproteinase matrix metalloproteinase (MMP)-2 and MMP-9. miR-373 was downregulated in glioma samples and cell lines and suppressed malignancy in glioblastoma cells. HOXA-AS2 bound to miR-373 and negatively regulated its expression. Epidermal growth factor receptor (EGFR), a target of miR-373, increased the expression levels of VE-cadherin, as well as the expression and activity levels of MMP-2 and MMP-9, via activating phosphatidylinositol 3-kinase/serine/threonine kinase pathways. HOXA-AS2 knockdown combined with miR-373 overexpression yielded optimal tumor suppressive effects and the lowest VM density in vivo. CONCLUSION: HOXA-AS2 knockdown inhibited malignant glioma behaviors and VM formation via the miR-373/EGFR axis.

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

BACKGROUND: Accumulating 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. RESULTS: Real-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. CONCLUSION: This 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.

Transcription Factor NFAT5 Promotes Glioblastoma Cell-driven Angiogenesis via SBF2-AS1/miR-338-3p-Mediated EGFL7 Expression Change.

Glioblastoma (GBM) is the most aggressive primary intracranial tumor of adults and confers a poor prognosis due to high vascularization. Hence anti-angiogenic therapy has become a promising strategy for GBM treatment. In this study, the transcription factor nuclear factor of activated T-cells 5 (NFAT5) was significantly elevated in glioma samples and GBM cell lines, and positively correlated with glioma WHO grades. Knockdown of NFAT5 inhibited GBM cell-driven angiogenesis. Furthermore, long non-coding RNA SBF2 antisense RNA 1 (SBF2-AS1) was upregulated in glioma samples and knockdown of SBF2-AS1 impaired GBM-induced angiogenesis. Downregulation of NFAT5 decreased SBF2-AS1 expression at transcriptional level. In addition, knockdown of SBF2-AS1 repressed GBM cell-driven angiogenesis via enhancing the inhibitory effect of miR-338-3p on EGF like domain multiple 7 (EGFL7). In vivo study demonstrated that the combination of NFAT5 knockdown and SBF2-AS1 knockdown produced the smallest xenograft volume and the lowest microvessel density. NFAT5/SBF2-AS1/miR-338-3p/EGFL7 pathway may provide novel targets for glioma anti-angiogenic treatment.

The Role of HOTAIR/miR-148b-3p/USF1 on Regulating the Permeability of BTB.

Homeobox transcript antisense intergenic RNA (HOTAIR), as a long non-coding RNA (lncRNA), has been considered to play critical roles in the biological properties of various tumors. The purposes of this study were to investigate the role and possible molecular mechanisms of HOTAIR in regulating the permeability of blood tumor barrier (BTB) in vitro. Our present study elucidated that the expressions of HOTAIR and upstream stimulatory factor 1 (USF1) was up-regulated, but miR-148b-3p was down-regulated in glioma microvascular endothelial cells (GECs). Knockdown of HOTAIR could increase the permeability of BTB as well as down-regulated the expressions of tight junction related proteins ZO-1, occludin, claudin-5, but up-regulated miR-148b-3p expressions in GECs. Meanwhile, dual-luciferase reporter assays demonstrated that HOTAIR was a target RNA of miR-148b-3p. Furthermore, overexpression of miR-148b-3p increased the permeability of BTB by down-regulating the expressions of tight junction related proteins and USF1 in GECs, and vice versa. And further result revealed USF1 was a target of miR-148b-3p. Silence of USF1 increased the permeability of BTB duo to their interaction with the promoters of ZO-1, occludin, and claudin-5 in GECs. Taken together, our finding indicated that knockdown of HOTAIR increased BTB permeability via binding to miR-148b-3p, which further reducing tight junction related proteins in GECs by targeting USF1. Thus, HOTAIR will attract more attention since it can serve as a potential target of drug delivery across BTB and may provide novel strategies for glioma treatment.

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.

Autophagy Induction by Endothelial-Monocyte Activating Polypeptide II Contributes to the Inhibition of Malignant Biological Behaviors by the Combination of EMAP II with Rapamycin in Human Glioblastoma.

This study aims to investigate the effect of endothelial-monocyte activating polypeptide II (EMAP II) on human glioblastoma (GBM) cells and glioblastoma stem cells (GSCs) as well as its possible mechanisms. In this study, EMAP II inhibited the cell viability and decreased the mitochondrial membrane potential in human GBM cells and GSCs, and autophagy inhibitor 3-methyl adenine (3-MA) blocked these effects. Autophagic vacuoles were formed in these cells after EMAP II treatment and this phenomenon was blocked by 3-MA. In addition, the up-regulation of microtubule-associated protein-1 light chain-3 (LC3)-II and the down-regulation of autophagic degraded substrate p62/SQSTM1 caused by EMAP II were observed. Cells treated with EMAP-II inhibited the PI3K/Akt/mTOR signal pathway, and PI3K/Akt agonist insulin-like growth factor-1 (IGF-1) blocked the effect of EMAP II on the expression of LC3-II and p62/SQSTM1. Cells exposed to EMAP-II experienced mitophagy and ER stress. Furthermore, the inhibition of cell proliferation, migration and invasion of GBM cells and GSCs were more remarkable by the combination of EMAP II and rapamycin than either agent alone in vitro and in vivo. The current study demonstrated that the cytotoxicity of EMAP II in human GBM cells and GSCs was induced by autophagy, accompanied by the inhibition of PI3K/Akt/mTOR signal pathway, mitophagy and ER stress. The combination of EMAP II with rapamycin demonstrated the inhibitory effect on the malignant biological behaviors of human GBM cells and GSCs in vitro and in vivo.

Overexpression of miR-18a negatively regulates myocyte enhancer factor 2D to increase the permeability of the blood-tumor barrier via Krüppel-like factor 4-mediated downregulation of zonula occluden-1, claudin-5, and occludin.

miR-18a represses angiogenesis and tumor evasion by weakening vascular endothelial growth factor and transforming growth factor-ß signaling to prolong the survival of glioma patients, although it is thought to be an oncogene. This study investigates the potential effects of miR-18a on the permeability of the blood-tumor barrier (BTB) and its possible molecular mechanisms. An in vitro BTB model was successfully established. The endogenous expression of miR-18a in glioma vascular endothelial cells (GECs) was significantly lower than that in normal vascular ECs, and the overexpression of miR-18a significantly increased the permeability of the BTB as well as downregulating the mRNA and protein expressions of tight junction-related proteins zonula occluden-1 (ZO-1), claudin-5, and occludin in GECs. Dual luciferase reporter assays revealed that miR-18a bound to the 3'-untranslated region (3'UTR) of myocyte enhancer factor 2D (MEF2D). The overexpression of both miR-18a and MEF2D with the 3'UTR significantly weakened the effect caused by miR-18a of decreasing the mRNA and protein expressions of ZO-1, claudin-5 and occludin and of increasing the permeability of the BTB. Chromatin immunoprecipitation showed that MEF2D could directly bind to KLF4 promoter. This study shows that miR-18a targets and negatively regulates MEF2D, which further regulates tight junction-related proteins ZO-1, claudin-5, and occludin through transactivation of KLF4 and, finally, changes the permeability of the BTB. MiR-18a should garner growing attention because it might serve as a potential target in opening the BTB and providing a new strategy for the treatment of gliomas.

MiR-18a increased the permeability of BTB via RUNX1 mediated down-regulation of ZO-1, occludin and claudin-5.

The purposes of this study were to investigate the possible molecular mechanisms of miR-18a regulating the permeability of blood-tumor barrier (BTB) via down-regulated expression and distribution of runt-related transcription factor 1 (RUNX1). An in vitro BTB model was established with hCMEC/D3 cells and U87MG cells to obtain glioma vascular endothelial cells (GECs). The endogenous expressions of miR-18a and RUNX1 were converse in GECs. The overexpression of miR-18a significantly impaired the integrity and increased the permeability of BTB, which respectively were detected by TEER and HRP flux assays, accompanied by down-regulated mRNA and protein expressions and distributions of ZO-1, occludin and claudin-5 in GECs. Dual-luciferase reporter assay was carried out and revealed RUNX1 is a target gene of miR-18a. Meanwhile, mRNA and protein expressions and distribution of RUNX1 were downregulated by miR-18a. Most important, miR-18a and RUNX1 could reversely regulate the permeability of BTB as well as the expressions and distributions of ZO-1, occludin and claudin-5. Finally, chromatin immunoprecipitation verified that RUNX1 interacted with "TGGGGT" DNA sequence in promoter region of ZO-1, occludin and claudin-5 respectively. Taken together, our present study indicated that miR-18a increased the permeability of BTB via RUNX1 mediated down-regulation of tight junction related proteins ZO-1, occludin and claudin-5, which would attract more attention to miR-18a and RUNX1 as potential targets of drug delivery across BTB and provide novel strategies for glioma treatment.

miR-34c regulates the permeability of blood-tumor barrier via MAZ-mediated expression changes of ZO-1, occludin, and claudin-5.

The purposes of this study were to investigate the potential roles of miR-34c in regulating blood-tumor barrier (BTB) functions and its possible molecular mechanisms. The over-expression of miR-34c significantly impaired the integrity and increased the permeability of BTB, which were detected in an in vitro BTB model by transendothelial electric resistance and horseradish peroxidase flux assays, respectively. Meanwhile, real-time quantitative PCR (qRT-PCR), Western blot and immunofluorescence assays successively demonstrated downregulation of ZO-1, occludin, and claudin-5 and miR-34c silencing uncovered the opposite results. Dual-luciferase reporter assays results revealed myc-associated zinc-finger protein (MAZ) is a target gene of miR-34c. Besides, mRNA and protein expressions of MAZ were reversely regulated by miR-34c. The down-expression of MAZ significantly impaired the integrity and increased the permeability of BTB as well as downregulated the expressions of ZO-1, occludin, and claudin-5. And chromatin immunoprecipitation verified that MAZ interacted with "GGGCGGG," "CCCTCCC," and "GGGAGGG" DNA sequence of ZO-1, occludin, and claudin-5 promoter, respectively. The over-expression or silencing of either miR-34c or MAZ was performed simultaneously to further explore their functional relations, and results elucidated that miR-34c and MAZ displayed reverse regulatory effects on the integrity and permeability of BTB as well as the expressions of ZO-1, occludin, and claudin-5. In conclusion, our present study indicated that miR-34c regulated the permeability of BTB via MAZ-mediated expression changes of ZO-1, occludin, and claudin-5.