CircMTA1 promotes glioblastoma angiogenesis by encoding MTA1-134aa.

Glioblastoma multiforme (GBM) is the most malignant brain tumor with rapid angiogenesis. How to inhibit GBM angiogenesis is a key problem to be solved. To explore the targets of inhibiting GBM angiogenesis, this study confirmed that the expression of circMTA1 (hsa_circ_0033614) was significantly upregulated in human brain microvascular endothelial cells exposed to glioma cell-conditioned medium (GECs). The expression of circMTA1 in the cytoplasm was significantly higher than that in the nucleus. Upregulated circMTA1 in GECs can promote cell proliferation, migration, and tube formation. Further exploration of the circularization mechanism of circMTA1 confirmed that KHDRBS1 protein can bind to the upstream and downstream flanking sequences of circMTA1 and promote circMTA1 biogenesis by coordinating Alu element pairing. KHDRBS1 upregulated the proliferation, migration, and tube formation of GECs by promoting the biogenesis of circMTA1. CircMTA1 can encode the protein MTA1-134aa by internal ribosome entry site sequence-mediated translation mechanism, and promote the proliferation, migration, and tube formation of GECs through the encoded MTA1-134aa. This study provides a new target for inhibiting angiogenesis in brain GBM and a new strategy for improving the therapeutic efficacy of GBM.

Metformin Reduces the Senescence of Renal Tubular Epithelial Cells in Diabetic Nephropathy via the MBNL1/miR-130a-3p/STAT3 Pathway.

Senescence of renal tubular epithelial cells plays an important role in diabetic nephropathy, but the mechanism is unknown. Metformin may alleviate diabetic nephropathy by reducing this senescence. This study is aimed at clarifying the effects and mechanism of metformin on the senescence of renal tubular epithelial cells in diabetic nephropathy. We found that metformin reduced the expression of senescence-associated gene P21 in high-glucose-induced (30 mmol/L) renal tubular epithelial cells and decreased the ß-galactosidase positive staining rate (decreased 16%, p < 0.01). Metformin was able to reduce senescence by upregulating the expression of RNA-binding protein MBNL1 and miR-130a-3p and reducing STAT3 expression. MBNL1 prolonged the half-life of miR-130a-3p, and miR-130a-3p could negatively regulate STAT3 by binding to its mRNA 3'UTR. In db/db diabetic mice, we found an enhanced senescence level combined with low expression of MBNL1 and miR-130a-3p and high expression of STAT3 compared with db/m control mice during nephropathy development. Meanwhile, metformin (200 mg/kg/day) could increase the expression of MBNL1 and miR-130a-3p and decreased STAT3 expression, thus reducing this senescence in db/db mice. Our results suggest that metformin reduces the senescence of renal tubular epithelial cells in diabetic nephropathy via the MBNL1/miR-130a-3p/STAT3 pathway, which provided new ideas for the therapy of this disease.

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.

Long Non-coding RNA TUSC7, a Target of miR-23b, Plays Tumor-Suppressing Roles in Human Gliomas.

Tumour suppressor candidate 7 (TUSC7) is a novel tumor suppressor gene generating long non-coding RNA (lncRNAs) in several types of human cancers. The expression and function of TUSC7 in human brain glioma has yet to be elucidated. In this study, TUSC7 was poorly expressed in tissues and cell lines of glioma, and the lower expression was correlated with glioma of the worse histological grade. Moreover, TUSC7 is a prognostic biomarker of glioma patients. Up-regulation of TUSC7 suppressed cellular proliferation and invasion of glioma cells, and accelerated cellular apoptosis. Bioinformatics analysis showed that TUSC7 specifically binds to miR-23b. MiR-23b was up-regulated in glioma and negatively correlated with the expression of TUSC7. The miR-23b expression was inhibited remarkably by the upregulation of TUSC7 and the reciprocal inhibition was determined between TUSC7 and miR-23b.RNA pull-down and luciferase reporter assays were used to validate the sequence-specific correlation between miR-23b and TUSC7. TUSC7 inhibited the proliferation, migration and invasion of glioma cells and promoted cellular apoptosis largely bypassing miR-23b. We conclude that the lncRNA TUSC7 acted as a tumor suppressor gene negatively regulated by miR-23b, suggesting a novel therapeutic strategy against gliomas.

NS1619 regulates the expression of caveolin-1 protein in a time-dependent manner via ROS/PI3K/PKB/FoxO1 signaling pathway in brain tumor microvascular endothelial cells.

NS1619, a calcium-activated potassium channel (Kca channel) activator, can selectively and time-dependently accelerate the formation of transport vesicles in both the brain tumor capillary endothelium and tumor cells within 15min of treatment and then increase the permeability of the blood-brain tumor barrier (BTB). However, the mechanism involved is still under investigation. Using a rat brain glioma (C6) model, the expression of caveolin-1, FoxO1 and p-FoxO1 protein were examined at different time points after intracarotid infusion of NS1619 at a dose of 30µg/kg/min. Internalization of Cholera toxin subunit (CTB) labeled fluorescently was monitored by flow cytometry. The expression of caveolin-1 and FoxO1 protein at tumor microvessels was enhanced and caveolae-mediated CTB endocytosis was increased by NS1619 infusion for 15min. Compared with the 15min group, the expression of caveolin-1 protein was significantly decreased and the level of phosphorylation of FoxO1 was significantly increased in the NS1619 2h group. In addition, inhibitors of reactive oxygen species (ROS) or PI3K or PKB significantly attenuated the level of FoxO1 phosphorylation and also increased the expression of caveolin-1 protein in Human Brain Microvascular Endothelial Cells (HBMECs) cocultured with human glioma cells (U87) 2h after NS1619 treatment. This led to the conclusion that NS1619-mediated transport vesicle increase is, at least partly, related to the ROS/PI3K/PKB/FoxO1 signaling pathway.

Mir-338-3p Inhibits Malignant Biological Behaviors of Glioma Cells by Targeting MACC1 Gene.

BACKGROUND Human brain glioma is the most common endocranial tumor; its mortality and morbidity are very high. The objective of this study was to determine whether miR-338-3p can regulate malignant biological behaviors of glioma cells by targeted silencing of MACC1. MATERIAL AND METHODS The expression of miR-338-3p was detected by quantitative real-time PCR in brain glioma tissues and cell lines. Bioinformatics software was used to predict some potential target genes of miR-338-3p. Luciferase activities assay was used to verify the combination between target genes and miR-338-3p. And MACC1 protein expression was detected by Western blot. The apoptosis and proliferation ability were analyzed by MTT and flow cytometry assay. RESULTS Compared with normal brain tissues and cells, miR-338-3p in glioma tissues and cell lines was confirmed to be expressed at low levels, and down-regulation of miR-338-3p tended to be correlated with worse histological grade. Up-regulation of miR-338-3p promoted apoptosis and sharply inhibited cell proliferation ability of U251 and U87 cells. The luciferase activities assay, biotin-avidin pull-down assay, and western blot analysis verified that MACC1 was a specific target gene of miR-338-3p. Subsequent experiments found that up-regulation of MACC1 significantly inhibited the apoptosis and increased the cell proliferation ability of U251 and U87 cells. The regulation effects of miR-338-3p on malignant biological behaviors of glioma cells can be partly reversed by up-regulation of MACC1. CONCLUSIONS Down-regulation of miR-338-3p was an independent prognostic biomarker associated with poor prognosis in glioma patients; miR-338-3p acted as a tumor-suppressing gene whose silencing can inhibit malignant biological behaviors of glioma cells. MACC1 was a specific target gene of miR-338-3p, which regulates malignant biological behaviors of glioma cells partly through directly silencing MACC1 expression.

miR-128 regulates the apoptosis and proliferation of glioma cells by targeting RhoE.

In this study, we investigate whether miR-128 is capable of regulating the apoptosis and proliferation of human U251 glioma cells by downregulating RhoE. The expression of miR-128 was assessed by quantitative polymerase chain reaction in normal brain tissue and glioma samples. A significant downregulation of the expression of miR-128 was detected in glioma in contrast to normal brain tissue. Following the transfection of pre-miR-128 and anti-miR-128 into U251 cells, the high expression of miR-128 could inhibit proliferation and induce apoptosis in U251 cells, and those effects could be restored by miR-128 knockdown. To analyze the regulation mechanism of miR-128, TargetScan, miRanda and PicTar were used to ascertain whether RhoE was a potential target gene. Next, luciferase activity assay and western blot analysis confirmed that RhoE was a direct and specific target gene of miR-128. The advanced effects of pre-miR-128 on the apoptosis and proliferation of U251 cells were reversed by the upregulation of RhoE expression. In summary, aberrantly expressed miR-128 regulates apoptosis and proliferation in human glioma U251 cells partly by directly targeting RhoE. This finding may offer a new potential therapeutic strategy for the treatment of glioma.

DT(270-326) , a Truncated Diphtheria Toxin, Increases Blood-Tumor Barrier Permeability by Upregulating the Expression of Caveolin-1.

AIM: The nontoxic mutant of diphtheria toxin (DT) has been demonstrated to act as a receptor-specific carrier protein to delivery drug into brain. Recent research showed that the truncated "receptorless" DT was still capable of being internalized into cells. This study investigated the effects and potential mechanisms of DT(270-326) , a truncated "receptorless" DT, on the permeability of the blood-tumor barrier (BTB). METHODS: BTB and GECs were subjected to DT(270-326) treatment. HRP flux assays, immunofluorescent, co-immunoprecipitation, Western blot, CCK-8, and Flow cytometry analysis were used to evaluate the effects of DT(270-326) administration. RESULTS: Our results revealed that 5 µM of DT(270-326) significantly increased the permeability of BTBin vitro, which reached its peak at 6 h. The permeability was reduced by pretreatment with filipinIII. DT(270-326) co-localized and interacted with caveolin-1 via its caveolin-binding motif. The mRNA and protein expression levels of caveolin-1 were identical with the changes of BTB permeability. The upregulated expression of caveolin-1 was associated with Src kinase-dependent tyrosine phosphorylation of caveolin-1, which subsequently induced phosphorylation and inactivation of the transcription factor Egr-1. The combination of DT(270-326) with doxorubicin significantly enhanced the loss of cell viability and apoptosis of U87 glioma cells in contrast to doxorubicin alone. CONCLUSIONS: DT(270-326) might provide a novel strategy to increase the delivery of macromolecular therapeutic agents across the BTB.

MiRNA-200b Regulates RMP7-Induced Increases in Blood-Tumor Barrier Permeability by Targeting RhoA and ROCKII.

The primary goals of this study were to investigate the potential roles of miR-200b in regulating RMP7-induced increases in blood-tumor barrier (BTB) permeability and some of the possible molecular mechanisms associated with this effect. Microarray analysis revealed 34 significantly deregulated miRNAs including miR-200b in the BTB as induced by RMP7 and 8 significantly up-regulated miRNAs in the BTB by RMP7. RMP7 induced tight junction (TJ) opening of the BTB, thereby increasing BTB permeability. Associated with this effect of RMP7 was a decrease in miR-200b expression within the human cerebral microvascular endothelial cells line hCMEC/D3 (ECs) of the BTB. Overexpression of miR-200b inhibited endothelial leakage and restored normal transendothelial electric resistance values. A simultaneous shift in occludin and claudin-5 distributions from insoluble to soluble fractions were observed to be significantly reduced. In addition, overexpression of miR-200b inhibited the relocation of occludin and claudin-5 from cellular borders into the cytoplasm as well as the production of stress fiber formation in GECs (ECs with U87 glioma cells co-culturing) of the BTB. MiR-200b silencing produced opposite results as that obtained from that of the miR-200b overexpression group. Overexpression of miR-200b was also associated with a down-regulation in RhoA and ROCKII expression, concomitant with a decrease in BTB permeability. Again, results which were opposite to that obtained with the miR-200b silencing group. We further found that miR-200b regulated BTB permeability by directly targeting RhoA and ROCKII. Collectively, these results suggest that miR-200b's contribution to the RMP7-induced increase in BTB permeability was associated with stress fiber formation and TJ disassembly as achieved by directly targeting RhoA and ROCKII.

Long noncoding RNA HOTAIR is a prognostic biomarker and inhibits chemosensitivity to doxorubicin in bladder transitional cell carcinoma.

PURPOSE: In this study, whether HOTAIR is a prognostic biomarker will be detected, and its regulative effects of chemosensitivity to doxorubicin in TCC cells will be examined. METHODS: The expression of HOTAIR was detected by quantitative real-time PCR. Overall survival rate was calculated by Kaplan-Meier method with the log-rank test for comparisons. MTT assay was used to detect cell proliferation ability and chemosensitivity. Dual-color flow cytometric method was used to detect cell apoptosis. RESULTS: HOTAIR was up-regulated in bladder transitional cell carcinoma (TCC) tissues and cell lines compared with normal bladder transitional cell (NBTC) tissues and bladder epithelial immortalized SV-HUC-1 cells, and its expression level had positive correlation with histological grades of TCC. Moreover, HOTAIR was an independent prognostic biomarker of overall survival for TCC patients. The expression and silence vector for HOTAIR were transfected into T24 and J82 cells to up-regulate and silence the HOTAIR expression, respectively. In T24 and J82 cells, HOTAIR over-expression promoted cell proliferation and inhibited chemosensitivity to doxorubicin and cell apoptosis induced by doxorubicin; silence of HOTAIR showed opposite regulative effects. CONCLUSIONS: In summary, lncRNA HOTAIR was an independent prognostic biomarker of overall survival in TCC patients and could regulate chemosensitivity to doxorubicin of human TCC cells. HOTAIR might provide a new potential therapeutic target and stratagem for TCC.

Low-Dose Endothelial Monocyte-Activating Polypeptide-II Induces Blood-Tumor Barrier Opening Via the cAMP/PKA/Rac1 Pathway.

Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood-tumor barrier (BTB) hyperpermeability via both paracellular and transcellular pathways. In a recent study, we revealed that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent signaling pathway is involved in EMAP-II-induced BTB hyperpermeability. This study further investigated the exact mechanisms through which the cAMP/PKA-dependent signaling pathway affects EMAP-II-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II (0.05 nM) induced a significant decrease in Rac1 activity in rat brain microvascular endothelial cells (RBMECs). Pretreatment with forskolin to elevate intracellular cAMP concentration completely blocked EMAP-II-induced inactivation of Rac1. Besides, pretreatment with 6Bnz-cAMP to activate PKA partially attenuated EMAP-II-induced Rac1 inactivation. Moreover, 6Bnz-cAMP pretreatment significantly diminished EMAP-II-induced changes in BTB permeability, myosin light chain (MLC) phosphorylation, expression and distribution of ZO-1, and actin cytoskeleton arrangement in RBMECs. These effects of 6Bnz-cAMP were completely blocked in the presence of NSC-23766 (the specific inhibitor of Rac1). In conclusion, this study demonstrates that low-dose EMAP-II induces BTB hyperpermeability via the cAMP/PKA/Rac1 signaling pathway.

Low-Dose Endothelial Monocyte-Activating Polypeptide-II Increases Blood-Tumor Barrier Permeability by Activating the RhoA/ROCK/PI3K Signaling Pathway.

Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can increase blood-tumor barrier (BTB) permeability via both paracellular and transcellular pathways. In addition, we revealed that the RhoA/Rho kinase (ROCK) signaling pathway is involved in EMAP-II-induced BTB opening. This study further investigated the exact mechanisms by which the RhoA/ROCK signaling pathway affects EMAP-II-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II significantly activated phosphatidylinositol-3-kinase (PI3K) in rat brain microvascular endothelial cells (RBMECs) at 0.75 h. Pretreatment with RhoA inhibitor C3 exoenzyme or ROCK inhibitor Y-27632 completely blocked EMAP-II-induced activation of PI3K. PKC-α/ß inhibitor GÖ6976 pretreatment caused no change in EMAP-II-induced activation of PI3K. Besides, pretreatment with LY294002, a specific inhibitor of PI3K, did not affect EMAP-II-induced activation of PKC-α/ß. Furthermore, LY294002 pretreatment significantly diminished EMAP-II-induced changes in BTB permeability, phosphorylation of myosin light chain and cofilin, expression and distribution of tight junction-associated protein ZO-1, and actin cytoskeleton arrangement in RBMECs. In summary, this study demonstrates that low-dose EMAP-II can increase BTB permeability by activating the RhoA/ROCK/PI3K signaling pathway.

MiR-143 enhances the antitumor activity of shikonin by targeting BAG3 expression in human glioblastoma stem cells.

Therapeutic applications of microRNAs (miRNAs) in chemotherapy were confirmed to be valuable, but there is rare to identify their specific roles and functions in shikonin treatment toward tumors. Here, for the first time, we reported that miR-143 played a critical role in the antitumor activity of shikonin in glioblastoma stem cells (GSCs). The results showed that the expression of miR-143 was downregulated in shikonin treated GSCs within 24 h. MiR-143 overexpression significantly enhanced the inhibitory effect of shikonin toward GSCs on cell viability. Besides, miR-143 overexpression caused a significant increase in the apoptotic fraction and made apoptosis occur earlier. Further investigation identified that BAG3, an apoptotic regulator, was a functional target of miR-143 in shikonin treated GSCs. The expression of BAG3 was upregulated in shikonin treated GSCs within 24 h. MiR-143 overexpression significantly reversed the high expression of BAG3 in shikonin treated GSCs. Moreover, it was confirmed that the enhanced cytotoxicity of shikonin by miR-143 overexpression was reversed by BAG3 overexpression both in vitro and in vivo, suggesting that the enhanced tumor suppressive effects by miR-143 overexpression was at least partly through the regulation of BAG3. Taken together, for the first time, our results demonstrate that miR-143 could enhance the antitumor activity of shikonin toward GSCs through reducing BAG3 expression, which may provide a novel therapeutic strategy for enhancing the treatment efficacy of shikonin toward GSCs.

Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated ß-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine.

Shikonin is an anthraquinone derivative extracted from the root of lithospermum. Shikonin is traditionally used in the treatment of inflammatory and infectious diseases such as hepatitis. Shikonin also inhibits proliferation and induces apoptosis in various tumors. However, the effect of shikonin on gliomas has not been fully elucidated. In the present study, we aimed to investigate the effects of shikonin on the migration and invasion of human glioblastoma cells as well as the underlying mechanisms. U87 and U251 human glioblastoma cells were treated with shikonin at 2.5, 5, and 7.5 µmol/L and cell viability, migration and invasiveness were assessed with CCK8, scratch wound healing, in vitro Transwell migration, and invasion assays. The expression and activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and the expression of phosphorylated ß-catenin (p-ß-catenin) and phosphorylated PI3K/Akt were also checked. Results showed that shikonin significantly inhibited the cell proliferation, migration, invasion, and expression of MMP-2 and MMP-9 in U87 and U251 cells. The expression of p-ß-catenin showed contrary trends in two cell lines. It was significantly inhibited in U87 cells and promoted in U251 cells. Results in this work indicated that shikonin displayed an inhibitory effect on the migration and invasion of glioma cells by inhibiting the expression and activity of MMP-2 and -9. In addition, shikonin also inhibited the expression of p-PI3K and p-Akt to attenuate cell migration and invasion and MMP-2 and MMP-9 expression in both cell lines, which could be reversed by the PI3K/Akt pathway agonist, insulin-like growth factor-1 (IGF-1).

Enhanced antitumor effect of shikonin by inhibiting Endoplasmic Reticulum Stress via JNK/c-Jun pathway in human glioblastoma stem cells.

Though previous study demonstrated that shikonin could exert its antitumor activity by inducing apoptosis and necrosis, the pro-survival mechanisms involved in its antitumor process are still little to know. In the present study, for the first time, we found a protective mechanism was simultaneously activated which caused the reduced sensitivity of glioblastoma stem cells (GSCs) to the cytotoxicity of shikonin. Reduced active caspase-9 expression and enhanced mitochondrial membrane potential (MMP) were intriguingly observed within 24 h treatment by shikonin in GSCs. Further investigation identified that Endoplasmic Reticulum Stress (ERS) was involved in its antitumor process, which compromised the cytotoxicity of shikonin toward GSCs. Inhibiting ERS by 4-phenylbutyric acid (4-PBA) markedly enhanced the cytotoxicity of shikonin in GSCs. The consistent result was simultaneously observed in the GSCs-xenografted mice. Furthermore, our results identified that JNK/c-Jun pathway was involved in the antitumor process of shikonin, providing a mechanism by which ERS reduced the cytotoxicity of shikonin toward GSCs. Altogether, the novel observation in the present study identified that inhibiting ERS would be an attractive new approach to enhance the therapeutic potency of shikonin toward GSCs.

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.

CRNDE affects the malignant biological characteristics of human glioma stem cells by negatively regulating miR-186.

The long non-coding RNA Colorectal neoplasia differentially expressed (CRNDE) is a novel gene that activated early in colorectal neoplasia, but it is also up-regulated in many other solid tumors. Herein, the function and underlying mechanism of CRNDE in regulating glioma stem cells (GSCs) were investigated. We found that CRNDE expression was up-regulated while miR-186 expression was down-regulated in GSCs. Overexpression of CRNDE could promote the cellular proliferation, migration, invasion and inhibit the apoptosis in GSCs. Overexpression of miR-186 exerted functions of inhibiting the proliferation, migration and invasion of GSCs and promoting apoptosis. And CRNDE decreased the expression levels of XIAP and PAK7 by binding to miR-186 and negatively regulating it. In addition, miR-186 binded to XIAP and PAK7 3'UTR region, and decrease the expression of them, thus regulating the expression levels of downstream target proteins such as caspase 3, BAD, cyclin D1 and MARK2. The in vivo effect of CRNDE and miR-186 showed that the tumor formation rate was minimum in tumor-bearing nude mice with the knockdown of CRNDE and the overexpression of miR-186. In conclusion, CRNDE played an oncogenic role of GSCs through the negative regulation of miR-186. Both CRNDE and miR-186 could be regarded as potential targets in the glioma therapy.

Knockdown of long non-coding RNA HOTAIR inhibits malignant biological behaviors of human glioma cells via modulation of miR-326.

Glioma is the most common and aggressive primary adult brain tumor. Long non-coding RNAs (lncRNAs) have important roles in a variety of biological properties of cancers. Here, we elucidated the function and the possible molecular mechanisms of lncRNA HOTAIR in human glioma U87 and U251 cell lines. Quantitative RT-PCR demonstrated that HOTAIR expression was up-regulated in glioma tissues and cell lines. Knockdown of HOTAIR exerted tumor-suppressive function in glioma cells. Further, HOTAIR was confirmed to be the target of miR-326 and miR-326 mediated the tumor-suppressive effects of HOTAIR knockdown on glioma cell lines. Moreover, over-expressed miR-326 reduced the FGF1 expression which played an oncogenic role in glioma by activating PI3K/AKT and MEK 1/2 pathways. In addition, the in vivo studies also supported the above findings. Taken together, knockdown of HOTAIR up-regulated miR-326 expression, and further inducing the decreased expression of FGF1, these results provided a comprehensive analysis of HOTAIR-miR-326-FGF1 axis in human glioma and provided a new potential therapeutic strategy for glioma treatment.

Functions for the cAMP/Epac/Rap1 Signaling Pathway in Low-Dose Endothelial Monocyte-Activating Polypeptide-II-Induced Opening of Blood-Tumor Barrier.

Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood-tumor barrier (BTB) hyperpermeability via both paracellular and transcellular pathways. In a recent study, we revealed that cAMP/PKA-dependent and cAMP/PKA-independent signaling pathways are both involved in EMAP-II-induced BTB hyperpermeability. The present study further investigated the exact mechanisms through which the cAMP/PKA-independent signaling pathway affects EMAP-II-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II (0.05 nM) induced a significant decrease in Rap1 activity in RBMECs. Pretreatment with forskolin to elevate intracellular cAMP concentration completely blocked EMAP-II-induced Rap1 inactivation. Epac/Rap1 activation by 8-pCPT-2'-O-Me-cAMP significantly prevented EMAP-II-induced activation of RhoA/ROCK. Furthermore, 8-pCPT-2'-O-Me-cAMP pretreatment significantly inhibited EMAP-II-induced decreases in TEER and increases in HRP flux. Pretreatment also significantly prevented EMAP-II-induced changes in MLC phosphorylation, actin cytoskeleton arrangement, and expression and distribution of ZO-1 in RBMECs. This study demonstrates that the cAMP/Epac/Rap1 signaling cascade is a crucial pathway in EMAP-II-induced BTB hyperpermeability.

Integrin α4 is involved in the regulation of glioma-induced motility of bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMSCs) have the ability of migrating towards glioma tissue. However, this migratory behavior remains to be elucidated. The aim of this study was to define the role of integrin α4 in the motility of BMSCs towards glioma. The role of integrin α4 in the migration of BMSCs towards glioma was evaluated using an in vitro migration assay with the application of a specific integrin α4­blocking antibody. The effect of glioma conditioned medium (CM) on the integrin α4 expression level of BMSCs was assessed by RT-PCR, immunocytochemistry and western blot analysis. BAY11-7082, LY294002, SB203580, PD98059 and SP600125 were used to investigate the role of NF-κB, PI3K, p38 MAPK, MEK and JNK in the above process. In addition, the role of NF-κB in the tropism of BMSCs towards glioma was also evaluated using the in vitro model. The migration of BMSCs towards glioma CM was attenuated by blocking integrin α4. The stimulation of glioma CM increased integrin α4 expression of BMSCs. Furthermore, the inhibition of NF-κB and PI3K decreased the glioma-induced integrin α4 upregulation on BMSCs. Inhibition of NF-κB decreased the number of migrating BMSCs towards gliomas. Glioma cells induced the migration of BMSCs by promoting the expression of integrin α4. NF-κB and PI3K contributed to the signal transduction of this process. Similar to PI3K, NF-κB is associated with the regulation of BMSCs migration toward glioma. Thus, these results may be useful to elucidate the mechanism involved in the glioma-induced migration of BMSCs.