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.

Expression profiles and function prediction of tRNA-derived fragments in glioma.

BACKGROUND: Glioblastoma (GBM) is the most aggressive malignant primary brain tumor. The transfer RNA-derived fragments (tRFs) are a new group of small noncoding RNAs, which are dysregulated in many cancers. Until now, the expression and function of tRFs in glioma remain unknown. METHODS: The expression profiles of tRF subtypes were analyzed using the Cancer Genome Atlas (TCGA)-low-grade gliomas (LGG)/GBM dataset. The target genes of tRFs were subjected to Gene Ontology, Kyoto Encyclopedia and Gene set enrichment analysis of Genes and Genomes pathway enrichment analysis. The protein-protein interaction enrichment analysis was performed by STRING. QRT-PCR was performed to detect the expressions of tRFs in human glioma cell lines U87, U373, U251, and human astrocyte cell line SVG p12. Western blot assay was used to detect to the expression of S100A11. The interaction between tRF-19-R118LOJX and S100A11 mRNA 3'UTR was detected by dual-luciferase reporter assay. The effects of tRF-19-R118LOJX, tRF-19-6SM83OJX and S100A11 on the glioma cell proliferation, migration and in vitro vasculogenic mimicry formation ability were examined by CCK-8 proliferation assay, EdU assay, HoloMonitor cell migration assay and tube formation assay, respectively. RESULTS: tRF-19-R118LOJX and tRF-19-6SM83OJX are the most differentially expressed tRFs between LGG and GBM groups. The functional enrichment analysis showed that the target genes of tRF-19-R118LOJX and tRF-19-6SM83OJX are enriched in regulating blood vessel development. The upregulated target genes are linked to adverse survival outcomes in glioma patients. tRF-19-R118LOJX and tRF-19-6SM83OJX were identified to suppress glioma cell proliferation, migration, and in vitro vasculogenic mimicry formation. The mechanism of tRF-19-R118LOJX might be related to its function as an RNA silencer by targeting the S100A11 mRNA 3'UTR. CONCLUSION: tRFs would become novel diagnostic biomarkers and therapeutic targets of glioma, and the mechanism might be related to its post-transcriptionally regulation of gene expression by targeting mRNA 3'UTR.

MiR-661 inhibits glioma cell proliferation, migration and invasion by targeting hTERT.

In this study, we analyzed the functional role of miR-661 in glioma cell proliferation, migration and invasion. We found that overexpression of miR-661 obviously suppressed the proliferation, migration and invasion of glioma cells. MiRNA target prediction algorithms implied that hTERT is a candidate target gene for miR-661. A fluorescent reporter assay confirmed that miR-661 could lead to hTERT gene silencing by recognizing and specifically binding to the predicted site of the hTERT mRNA 3' untranslated region (3'UTR) specifically. Furthermore, hTERT knockdown significantly decreased the growth and viability of glioma cells. These results indicate that miR-661 can inhibit glioma cell proliferation, migration and invasion by targeting hTERT.

Changes in the numbers of ribbon synapses and expression of RIBEYE in salicylate-induced tinnitus.

BACKGROUND: This study was performed to explore the mechanism underlying tinnitus by investigating the changes in the synaptic ribbons and RIBEYE expression in cochlear inner hair cells in salicylate-induced tinnitus. METHODS: C57BL/6J mice were injected with salicylate (350 mg/kg) for 10 days and grouped. Behavioral procedures were performed to assess whether the animals experienced tinnitus. The specific presynaptic RIBEYE protein and non-specific postsynaptic glutamate receptor 2&3 protein in basilar membrane samples were examined by immunofluorescent labeling. RT-PCR and Western blot assays were used to examine RIBEYE expression. Serial sections were used to build three-dimensional models using 3ds MAX software to evaluate the changes in the synaptic ribbons. RESULTS: The administration of salicylate increased false positives in the behavioral procedure from 3 d to 10 d. The membrane profiles of inner hair cells in all mice were intact. The number of synaptic ribbons in the salicylate group increased on the 7(th) d and decreased on the 9(th) and 10(th) d. mRNA and protein expression of RIBEYE were initially up-regulated and later down-regulated by injecting salicylate for 10 consecutive days. CONCLUSION: This change in the ribbon synapses of cochlear inner hair cells in salicylate-induced mice might serve as a compensatory mechanism in the early stages of ototoxicity and contribute to tinnitus later. The alteration of RIBEYE expression could be responsible for the changes in the morphology of ribbon synapses and for salicylate-induced tinnitus.

Comprehensive Profiles of mRNAs and miRNAs Reveal Molecular Characteristics of Multiple Organ Physiologies and Development in Pigs.

The pig (Sus scrofa) is not only an important livestock animal but also widely used as a biomedical model. However, the understanding of the molecular characteristics of organs and of the developmental skeletal muscle of the pig is severely limited. Here, we performed a comprehensive transcriptome profiling of mRNAs and miRNAs across nine tissues and three skeletal muscle developmental stages in the Guizhou miniature pig. The reproductive organs (ovary and testis) had greater transcriptome complexity and activity than other tissues, and the highest transcriptome similarity was between skeletal muscle and heart (R = 0.79). We identified 1,819 mRNAs and 96 miRNAs to be tissue-specific in nine organs. Testis had the largest number of tissue-specific mRNAs (992) and miRNAs (40). Only 15 genes and two miRNAs were specifically expressed in skeletal muscle and fat, respectively. During postnatal skeletal muscle development, the mRNAs associated with focal adhesion, Notch signaling, protein digestion, and absorption pathways were up-regulated from D0 to D30 and then down-regulated from D30 and D240, while genes with opposing expression patterns were significantly enriched in the oxidative phosphorylation and proteasome pathways. The miRNAs mainly regulated genes associated with insulin, Wnt, fatty acid biosynthesis, Notch, MAPK, TGF-beta, insulin secretion, ECM-receptor interaction, focal adhesion, and calcium signaling pathways. We also identified 37 new miRNA-mRNA interaction pairs involved in skeletal muscle development. Overall, our data not only provide a rich resource for understanding pig organ physiology and development but also aid the study of the molecular functions of mRNA and miRNA in mammals.

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.

Long non-coding RNA CASC2 suppresses malignancy in human gliomas by miR-21.

Long non-coding RNAs (lncRNAs) are aberrantly expressed in many diseases including cancer. LncRNA CASC2 (cancer susceptibility candidate 2) has been characterized as a tumor suppressor in endometrial cancer and colorectal cancer. However, the role and function of CASC2 in human gliomas remain unknown. In this study, we confirmed that CASC2 was lowly expressed in glioma tissues as well as in U251 and U87 glioma cell lines. Overexpression of CASC2 inhibited the malignancy of glioma cells, including proliferation, migration, and invasion, and promoted cell apoptosis. MicroRNA-21 (miR-21) has been reported to be overexpressed in human glioma tissues and cell lines, which is responsible for the malignant progression of glioma. We found that up-regulated CASC2 decreased the expression of miR-21 significantly and there is a reciprocal repression between CASC2 and miR-21 in an Argonaute2-dependent manner. Furthermore, bioinformatics, luciferase reporter assays and pull-down assay confirmed that miR-21 binds to CASC2 in a sequence-specific manner. Introduction of miR-21 largely abrogated CASC2-mediated inhibition of glioma cell proliferation, migration, and invasion, and promotion of cell apoptosis. This study demonstrated that CASC2 plays a tumor suppressive role in glioma via negative regulation of miR-21, which may be a novel therapeutic target for treating gliomas.

Roles of Serine/Threonine Phosphatases 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) opening via RhoA/Rho kinase/PKC-α/ß signaling pathway. In a recent study, we revealed that low-dose EMAP-II induced significant increases in expression levels of serine/threonine (Ser/Thr) phosphatase (PP)1 and 2A in rat brain microvascular endothelial cells (RBMECs) of BTB model. In addition, PKC-ζ/PP2A signaling pathway is involved in EMAP-II-induced BTB hyperpermeability. The present study further investigated the exact roles of PPs in this process. In an in vitro BTB model, low-dose EMAP-II (0.05 nM) induced a significant increase in PP1 activity in RBMECs. There was an interaction between PKC-α/ß and PP1 in RBMECs. Inhibition of PKC-α/ß activity with GÖ6976 completely blocked EMAP-II-induced activation of PP1. Conversely, inhibition of PP1 activity with tautomycin had no effect on EMAP-II-induced PKC-α/ß activation. Like GÖ6976, tautomycin significantly prevented EMAP-II-induced BTB hyperpermeability and MLC phosphorylation in RBMECs. Also, in this study, EMAP-II induced a marked redistribution of occludin and a significant dephosphorylation of occludin on Ser/Thr residues in RBMECs. Similar with GÖ6976 pretreatment, tautomycin pretreatment dramatically diminished EMAP-II-induced redistribution of occludin. Furthermore, pretreatment with tautomycin significantly inhibited EMAP-II-induced dephosphorylation of occludin on Ser residues. However, pretreatment with okadaic acid (an inhibitor of PP2A) significantly prevented changes in Ser-phosphorylated occludin induced by EMAP-II treatment. Collectively, this study demonstrates that low-dose EMAP-II increases BTB permeability via a RhoA/Rho kinase/PKC-α/ß/PP1 signaling pathway and that PP1/PP2A-mediated Ser/Thr dephosphorylation of occludin plays an important role in EMAP-II-induced BTB hyperpermeability.

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.

miR-101 acts as a tumor suppressor by targeting Kruppel-like factor 6 in glioblastoma stem cells.

BACKGROUND AND AIMS: Great interest persists in useful therapeutic targets in glioblastoma (GBM). Deregulation of microRNAs (miRNAs) expression has been associated with cancer formation through alterations in gene targets. In this study, we reported the role of miR-101 in human glioblastoma stem cells (GSCs) and the potential mechanisms. METHODS AND RESULTS: Quantitative real-time PCR showed that miR-101 expression was decreased in GSCs. Overexpression of miR-101 reduced the proliferation, migration, invasion, and promoted apoptosis of GSCs. One direct target of miR-101, the transcription factor Kruppel-like factor 6 (KLF6), was identified using the Dual-Luciferase Reporter Assay System, which mediated the tumor suppressor activity of miR-101. This process was coincided with the reduced expression of Chitinase-3-like protein 1 (CHI3L1) whose promoter could be bound with and be promoted by KLF6 demonstrated by luciferase assays and chromatin immunoprecipitation assays. The downregulation of CHI3L1 led to the inactivation of MEK1/2 and PI3K signal pathways. Furthermore, nude mice carrying the tumors of overexpressed miR-101 combined with knockdown of KLF6 produced the smallest tumors and showed the highest survival rate. CONCLUSIONS: Our findings provided a comprehensive analysis of miR-101 and further defining it as a potential therapeutic candidate for GBM.

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.

Artemether combined with shRNA interference of vascular cell adhesion molecule-1 significantly inhibited the malignant biological behavior of human glioma cells.

Artemether is the derivative extracted from Chinese traditional herb and originally used for malaria. Artemether also has potential therapeutic effects against tumors. Vascular cell adhesion molecule-1 (VCAM-1) is an important cell surface adhesion molecule associated with malignancy of gliomas. In this work, we investigated the role and mechanism of artemether combined with shRNA interference of VCAM-1 (shRNA-VCAM-1) on the migration, invasion and apoptosis of glioma cells. U87 human glioma cells were treated with artemether at various concentrations and shRNA interfering technology was employed to silence the expression of VCAM-1. Cell viability, migration, invasiveness and apoptosis were assessed with MTT, wound healing, Transwell and Annexin V-FITC/PI staining. The expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and phosphorylated Akt (p-Akt) was checked by Western blot assay. Results showed that artemether and shRNA-VCAM-1 not only significantly inhibited the migration, invasiveness and expression of MMP-2/9 and p-Akt, but also promoted the apoptosis of U87 cells. Combined treatment of both displayed the maximum inhibitory effects on the malignant biological behavior of glioma cells. Our work revealed the potential therapeutic effects of artemether and antiVCAM-1 in the treatments of gliomas.