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.

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.

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.

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.

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.

Bradykinin increased the permeability of BTB via NOS/NO/ZONAB-mediating down-regulation of claudin-5 and occludin.

After demonstrating bradykinin (BK) could increase the permeability of blood-tumor barrier (BTB) via opening the tight junction (TJ), and that the possible mechanism is unclear, we demonstrated that BK could increase the expressions of eNOS and nNOS and promote ZONAB translocation into nucleus. NOS inhibitors l-NAME and 7-NI could effectively block the effect of BK on increasing BTB permeability, decreasing the expressions of claudin-5 and occludin and promoting the translocation of ZONAB. Overexpression of ZONAB could significantly enhance BK-mediating BTB permeability. Meanwhile, chromatin immunoprecipitation verified ZONAB interacted with the promoter of claudin-5 and occludin respectively. This study indicated NOS/NO/ZONAB pathway might be involved in BK's increasing the permeability of BTB.

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.

Low-dose endothelial monocyte-activating polypeptide-II increases permeability of blood-tumor barrier via a PKC-ζ/PP2A-dependent signaling mechanism.

Our previous study demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood-tumor barrier (BTB) opening via the RhoA/Rho kinase/protein kinase C (PKC)-α/ß signaling pathway and that PKC-ζ is involved in this process via other mechanisms. In the present study, using an in vitro BTB model, we detected the exact signaling mechanisms by which PKC-ζ activation affects EMAP-II-induced BTB hyperpermeability. Our results showed that three types of serine/threonine (Ser/Thr) protein phosphatases (PPs), namely PP1, PP2A, and PP2B, were expressed by rat brain microvascular endothelial cells (RBMECs). There was an interaction between PKC-ζ and PP2A in RBMECs. In addition, EMAP-II induced a significant increase in both the expression and the activity of PP2A in RBMECs. Inhibition of PKC-ζ with PKC-ζ pseudosubstrate inhibitor (PKC-ζ-PI) completely blocked EMAP-II-induced PP2A activation. Conversely, inhibition of PP2A with okadaic acid (OA) had no effect on EMAP-II-induced PKC-ζ activation. Like PKC-ζ-PI, OA partially prevented EMAP-II-induced BTB hyperpermeability and occludin redistribution in RBMECs. Neither PKC-ζ-PI nor OA affected EMAP-II-induced phosphorylation of myosin light chain and redistribution of actin cytoskeleton in RBMECs. Taken together, our present study demonstrated that low-dose EMAP-II increases BTB permeability by activating the PKC-ζ/PP2A signaling pathway, which consequently leads to the disruption of TJs and impairment of endothelial barrier function.

Role of cAMP-dependent protein kinase A activity in low-dose endothelial monocyte-activating polypeptide-II-induced opening of blood-tumor barrier.

Our previous studies demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can selectively increase the permeability of blood-tumor barrier (BTB). In addition, low-dose EMAP-II significantly decreases the cyclic adenosine monophosphate (cAMP) concentration and the protein kinase A (PKA) expression level in tumor tissues in the rat C6 glioma model. In this study, an in vitro BTB model was used to investigate the potential role of cAMP/PKA signaling cascade in EMAP-II-induced BTB hyperpermeability. Our data revealed that low-dose EMAP-II (0.05 nM) induced a significant decrease in total intracellular cAMP concentration and PKA activity in rat brain microvascular endothelial cells (RBMECs). Pretreatment with forskolin to increase intracellular cAMP nearly completely blocked the EMAP-II-induced decrease in transendothelial electric resistance and increase in horseradish peroxidase flux across the BTB. Similar pretreatment completely prevented the EMAP-II-induced changes in RhoA/Rho kinase activity, expression and distribution of tight junction-associated protein ZO-1, and myosin light chain phosphorylation, as well as actin cytoskeleton arrangement in RBMECs. Pretreatment with 6Bnz-cAMP to activate PKA significantly attenuated these EMAP-II-induced alterations in RBMECs. In summary, our present study demonstrates that the cAMP/PKA signaling cascade works as a crucial signaling pathway in EMAP-II-induced BTB hyperpermeability.

Topoisomerase I inhibitors, shikonin and topotecan, inhibit growth and induce apoptosis of glioma cells and glioma stem cells.

Gliomas, the most malignant form of brain tumors, contain a small subpopulation of glioma stem cells (GSCs) that are implicated in therapeutic resistance and tumor recurrence. Topoisomerase I inhibitors, shikonin and topotecan, play a crucial role in anti-cancer therapies. After isolated and identified the GSCs from glioma cells successfully, U251, U87, GSCs-U251 and GSCs-U87 cells were administrated with various concentrations of shikonin or topotecan at different time points to seek for the optimal administration concentration and time point. The cell viability, cell cycle and apoptosis were detected using cell counting kit-8 and flow cytometer to observe the inhibitory effects on glioma cells and GSCs. We demonstrated that shikonin and topotecan obviously inhibited proliferation of not only human glioma cells but also GSCs in a dose- and time-dependent manner. According to the IC50 values at 24 h, 2 µmol/L of shikonin and 3 µmol/L of topotecan were selected as the optimal administration concentration. In addition, shikonin and topotecan induced cell cycle arrest in G0/G1 and S phases and promoted apoptosis. The down-regulation of Bcl-2 expression with the activation of caspase 9/3-dependent pathway was involved in the apoptosis process. Therefore, the above results showed that topoisomerase I inhibitors, shikonin and topotecan, inhibited growth and induced apoptosis of GSCs as well as glioma cells, which suggested that they might be the potential anticancer agents targeting gliomas to provide a novel therapeutic strategy.

Curcumin blocks small cell lung cancer cells migration, invasion, angiogenesis, cell cycle and neoplasia through Janus kinase-STAT3 signalling pathway.

Curcumin, the active component of turmeric, has been shown to protect against carcinogenesis and prevent tumor development. However, little is known about its anti-tumor mechanism in small cell lung cancer (SCLC). In this study, we found that curcumin can inhibit SCLC cell proliferation, cell cycle, migration, invasion and angiogenesis through suppression of the STAT3. SCLC cells were treated with curcumin (15 µmol/L) and the results showed that curcumin was effective in inhibiting STAT3 phosphorylation to downregulate of an array of STAT3 downstream targets ,which contributed to suppression of cell proliferation, loss of colony formation, depression of cell migration and invasion. Curcumin also suppressed the expression of proliferative proteins (Survivin, Bcl-X(L) and Cyclin B1), and invasive proteins (VEGF, MMP-2, MMP-7 and ICAM-1). Knockdown of STAT3 expression by siRNA was able to induce anti-invasive effects in vitro. In contrast, activation of STAT3 upstream of interleukin 6 (IL-6) leads to the increased cell proliferation ,cell survival, angiogenesis, invasion, migration and tumor growth. Our findings illustrate the biologic significance of IL-6/JAK/STAT3 signaling in SCLC progression and provide novel evidence that the pathway may be a new potential target for therapy of SCLC. It was concluded that curcumin is a potent agent in the inhibition of STAT3 with favorable pharmacological activity,and curcumin may have translational potential as an effective cancer therapeutic or preventive agent for SCLC.

[Pathological study of unexpected sudden death clustered in family or village in Yunnan province: report of 29 cases of autopsy].

OBJECTIVE: To investigate the pathological features and causes of sudden death clustered in family or village in Yunnan province so as to provide the morphological basis for exploring its etiology and medical intervention. METHODS: Autopsy was performed on 29 cases of clustered in family or village in Yunnan province during the period 1991-2006, 16 males and 13 females, aged 32 (8-69), accounting for 10.2% of whole sudden unexpected deaths occurring in the same period. The heart, lung, liver, spleen, brain, kidney, intestinal tract, and other organs were examined macroscopically and histologically, including a study of cardiac conduction system in 5 cases. Pathological diagnosis of myocarditis was based on the Dallas Criteria and World Heart Federation's consensus while the histological evaluation of Keshan disease referred the China national guideline for pathological diagnosis of Keshan disease. RESULTS: Based on the main pathological changes and the causes of death, these cases were classified into seven groups (group A-G). Group A comprised 11 cases (38%) with lymphocytic myocarditis accompanied with focal myocardial necrosis or degeneration. Group B comprised 3 cases (10%) with neutrophil myocarditis accompanied with focal myocytolysis or coagulation necrosis. Group C comprised 4 cases (14%) with arrhythmogenic right ventricle cardiomyopathy in which fatty infiltration of myocardium was the only pathological finding. Group D comprised 2 cases (7%) with ischemic heart disease in which fresh or old foci of myocardial infarction were found but coronary stenosis was shown only in one case. Group E comprised 2 cases (7%) with left ventricle hypertrophy and obstructive muscle bundle in the outflow of left ventricle. Group F comprised 2 cases (7%) with allergic bronchitis or chronic bronchitis and pulmonary emphysema. Group G comprised the remaining 5 cases (17%) without any pathological finding that could explain sudden death. No cases suffered with Keshan disease and dilated cardiomyopathy. Focal but not diffuse inflammatory infiltration was the prominent histological feature of myocarditis in Yunnan cases. Among the five cases with histological examination of cardiac conduction system, 2 cases were detected to suffer from acute hemorrhage in His bundle and its left branching site, and the atrioventricular node of 1 case was involved. Different pathological changes coexisted in 4 pairs of family members as a cluster of sudden deaths. 3 of 4 first deaths had focal myocarditis and the other one had chronic infection. But 3 secondary deaths had myocardial ischemia and the other one had arrhythmogenic right ventricle cardiomyopathy. Pulmonary edema, acute respiratory infection and congestive or ischemic liver necrosis were found in some cases simultaneously. CONCLUSION: The pathological changes of the cases of clustered sudden death in Yunnan province are various, such as myocarditis, myocardial dysplasia and the other lethal heart-lung disorders. No case of Keshan disease has been found. Arrhythmogenic right ventricle cardiomyopathy and other foundational heart diseases might act as a background. It is very hard to contribute only one etiological factor to the clustering of sudden death in Yunnan. It was most likely that multiple factors cluster and trigger an outbreak of death in a definite time and space.