Long noncoding RNA HNF1A-AS1 regulates proliferation and apoptosis of glioma through activation of the JNK signaling pathway via miR-363-3p/MAP2K4.

Long noncoding RNAs (lncRNAs) have been proven to exert important functions in the various biological processes of human cancers. It has been reported that lncRNA HNF1 homeobox A antisense RNA 1 (HNF1A-AS1) was abnormally expressed and played a role in the initiation and development of various human cancers. In this study, we confirmed that the expression level of HNF1A-AS1 was increased in glioma tissues and cells. Knockdown of HNF1A-AS1 inhibited cell proliferation and promoted cell apoptosis in glioma. Then, we disclosed the downregulation of miR-363-3p in glioma tissues and cell lines. The interaction between HNF1A-AS1 and miR-363-3p was identified in glioma cells. Furthermore, an inverse correlation between HNF1A-AS1 and miR-363-3p was observed in glioma tissues. Afterwards, we recognized that MAP2K4 was a direct target of miR-363-3p. The expression of MAP2K4 was negatively correlated with miR-363-3p while positively related to HNF1A-AS1 in glioma tissues. We also found the regulatory effect of HNF1A-AS1 on the MAP2K4-dependent JNK signaling pathway. All findings indicated that HNF1A-AS1 induces the upregulation of MAP2K4 to activate the JNK signaling pathway to promote glioma cell growth by acting as a miR-363-3p sponge.

HOXB-AS1 accelerates the tumorigenesis of glioblastoma via modulation of HOBX2 and HOBX3 at transcriptional and posttranscriptional levels.

Glioblastoma (GBM) is the most universal and invasive brain tumor among adults. Increasing studies have reported that long noncoding RNAs play vital roles in regulating downstream molecules at the transcriptional or posttranscriptional level in tumor progression. The purpose of the current research was to inquire the modulation mechanism by which homeobox B cluster antisense RNA 1 (HOXB-AS1) functioned in GBM. Our study first discovered the lifted expression of HOXB-AS1 and its nearby genes HOXB2 and HOXB3 in GBM and the positive relationship between HOXB-AS1 and HOXB2 or HOXB3. Loss-of-function assays and in vivo study detected that silencing of HOXB-AS1, HOXB2, or HOXB3 restrained the proliferation and induced the apoptosis in GBM. In addition, mechanism experiments demonstrated that HOXB-AS1 recruited interleukin enhancer-binding factor 3 (ILF3) to regulate HOXB2 and HOXB3 expression at the transcriptional level, and HOXB-AS1 sponged miR-186-5p to modulate HOXB2 and HOXB3 expression at posttranscriptional level. Finally, the regulatory mechanism of HOXB-AS1 in GBM was certified through rescue experiments. Our results indicated that HOXB-AS1 boost the HOXB2 or HOXB3 expression at the transcriptional and posttranscriptional levels. We detected the HOXB-AS1-ILF3-HOXB2/HOXB3 axis and HOXB-AS1-miR-186-5p-HOXB2/HOXB3 axis driving the GBM progression, which might generate more effective diagnostic biomarkers and therapeutic targets for patients with GBM.

ß1,6 GlcNAc branches-modified protein tyrosine phosphatase Mu attenuates its tyrosine phosphatase activity and promotes glioma cell migration through PLCγ-PKC pathways.

The metastatic potential of malignant tumor has been shown to be correlated with the increased expression of tri- and tetra-antennary ß1,6-N-acetylglucosamine (ß1,6-GlcNAc) N-glycans. In this study, We found that GnT-V expression was negatively correlated with receptor protein tyrosine phosphatase type µ(RPTPµ) in human glioma tissues. To study whether RPTPµ is a novel substance of GnT-V which further affect RPTPµ's downstream dephosphorylation function, we preform lentiviral infection with GnT-V gene to construct stably transfected GnT-V glial cell lines. We found RPTPµ undergone severer cleavage in GnT-V transfected glioma cells compare to Mock cells. RPTPµ intracellular domain fragments increased while ß1,6-GlcNAc-branched N-glycans increased, in consistent with the decrease of RPTPµ's catalytic activity. The results showed that abnormal glycosylation could decrease the phosphorylation activity of PTP µ, and affect PLCγ-PKC pathways. Both protease inhibitor Furin and N-glycan biosynthesis inhibitor swainsonine could decrease cell mobility in GnT-V-U87 transfectants and other glioma cell lines. All results above suggest increased post-translational modification of RPTPµ N-glycans by GnT-V attenuates its tyrosine phosphatase activity and promotes glioma cell migration through PLCγ-PKC pathways, and that the ß1,6-GlcNAc-branched N-glycans of RPTPµ play a crucial role in glioma invasivity.

N-Glycosylation at Asn 402 Stabilizes N-Cadherin and Promotes Cell-Cell Adhesion of Glioma Cells.

Cadherin is crucial for cell-cell adhesion and N-glycosylation of N-cadherin has been implicated in the process of mammary, renal, and ovarian carcinogenesis. However, whether N-glycosylation of N-cadherin plays a role in glioma remains unknown. Previous studies had indicated that N-glycosylation could occur at three asparagine residues of N-cadherin. By generating and over-expressing N-glycosylation-deficient N-cadherin mutants in the human glioma cell lines SHG66 and U87, we found that mutation of N402 but not of the other potentially N-glycosylated residues destabilized N-cadherin and led to its ubiquitylation and subsequent proteasomal degradation. Furthermore, destabilized N-cadherin inhibited cadherin-mediated cell-cell adhesion and promoted cell migration. Our findings reveal that N-glycosylation controls N-cadherin stability and plays a role in glioma migration. J. Cell. Biochem. 118: 1423-1431, 2017. © 2016 Wiley Periodicals, Inc.

The dual role of POSTN in maintaining glioblastoma stem cells and the immunosuppressive phenotype of microglia in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is an immunosuppressive, universally lethal cancer driven by glioblastoma stem cells (GSCs). The interplay between GSCs and immunosuppressive microglia plays crucial roles in promoting the malignant growth of GBM; however, the molecular mechanisms underlying this crosstalk are unclear. This study aimed to investigate the role of POSTN in maintaining GSCs and the immunosuppressive phenotype of microglia. METHODS: The expression of POSTN in GBM was identified via immunohistochemistry, quantitative real-time PCR, and immunoblotting. Tumorsphere formation assay, Cell Counting Kit-8 assay and immunofluorescence were used to determine the key role of POSTN in GSC maintenance. ChIP-seq and ChIP-PCR were conducted to confirm the binding sequences of ß-catenin in the promoter region of FOSL1. Transwell migration assays, developmental and functional analyses of CD4+ T cells, CFSE staining and analysis, enzyme-linked immunosorbent assays and apoptosis detection tests were used to determine the key role of POSTN in maintaining the immunosuppressive phenotype of microglia and thereby promoting the immunosuppressive tumor microenvironment. Furthermore, the effects of POSTN on GSC maintenance and the immunosuppressive phenotype of microglia were investigated in a patient-derived xenograft model and orthotopic glioma mouse model, respectively. RESULTS: Our findings revealed that POSTN secreted from GSCs promotes GSC self-renewal and tumor growth via activation of the αVß3/PI3K/AKT/ß-catenin/FOSL1 pathway. In addition to its intrinsic effects on GSCs, POSTN can recruit microglia and upregulate CD70 expression in microglia through the αVß3/PI3K/AKT/NFκB pathway, which in turn promotes Treg development and functionality and supports the formation of an immunosuppressive tumor microenvironment. In both in vitro models and orthotopic mouse models of GBM, POSTN depletion disrupted GSC maintenance, decreased the recruitment of immunosuppressive microglia and suppressed GBM growth. CONCLUSION: Our findings reveal that POSTN plays critical roles in maintaining GSCs and the immunosuppressive phenotype of microglia and provide a new therapeutic target for treating GBM.

Orthotopic model of SHG-44 in the enhanced green fluorescent protein nude mouse.

Naturally fluorescent proteins have been widely used in biological research. In this study, we found that the simple and effective way to obtain enhanced green fluorescent protein (EGFP) nude mice is to cross transgenic EGFP C57BL/6J mice with nude (nu/nu) mice. EGFP expression is identified by tail genotyping. Establishment of the orthotopic EGFP nude mouse model used surgical orthotopic implantation. The morphology and human glioma cell markers, such as glial fibrillary acidic protein (GFAP) and S-100, remain unchanged in this mouse model. The tumor blood vessels obtained from the orthotopic model show brilliant EGFP fluorescence as observed by fluorescence microscopy. These findings suggested that this is an ideal mouse model with which to study interaction among host, tumor, and tumor microenvironment; the findings also suggested that the host (EGFP nude mouse) was involved in tumor angiogenesis.

PSMB8-AS1 activated by ELK1 promotes cell proliferation in glioma via regulating miR-574-5p/RAB10.

Long noncoding RNAs (lncRNAs) get great involvements in the development of countless cancers. Nonetheless, the deep molecular mechanism by which lncRNA regulates the formation of glioma is unclear. In our study, the expression of PSMB8-AS1 was dramatically upregulated in glioma tissues and cells, further, PSMB8-AS1 silencing restrained cell proliferation in glioma, and the results of PSMB8-AS1 overexpression were opposite. Moreover, PSMB8-AS1 could bind with miR-574-5p, which was low expressed in glioma cells. In addition, RAB10 acted the target gene of miR-574-5p, and PSMB8-AS1 could regulate RAB10 via modulating miR-574-5p. Besides, miR-574-5p inhibitor/mimics remedied the repressive/simulative role of PSMB8-AS1 depletion/overexpression, and RAB10 downregulation/upregulation reversed the encouraging/blocked function caused by miR-574-5p inhibitor/mimics in PSMB8-AS1 depletion/overexpression transfected glioma cells. Additionally, ELK1, a transcription factor, could active PSMB8-AS1 expression. To be concluded, PSMB8-AS1 activated by ELK1 promotes cell proliferation in glioma via regulating miR-574-5p/RAB10, which may be contributory to find new targets to treat glioma.

RAD21 inhibited transcription of tumor suppressor MIR4697HG and led to glioma tumorigenesis.

BACKGROUND: Studies have revealed the aberrant expression of lncRNAs is responsible for human carcinogenesis. MIR4697 host gene (MIR4697HG) is an upregulated lncRNA that promoted cell growth and metastasis in other cancers. In this study, we tested the expression of MIR4697HG in glioma cells and detected the comparatively down-regulated expression. RAD1 is an upstream regulator for MIR4697HG. This study aimed at exploring the regulatory mechanism and function of RAD1/MIR4697HG/PRR12 axis in glioma. METHODS: We profiled the expression of MIR4697HG in glioblastoma multiforme (GBM) tissues according to GEPIA database as well as in glioma cells by qPCR. Functional experiments confirmed relevant role of MIR4697HG in regulating glioma cell proliferation and migration. We also carried out luciferase reporter assay, pull down assay and RIP assay to verify the location and interaction among the indicated RNA molecules. RESULTS: The expression of MIR4697HG is down-regulated significantly in glioma cells due to the up-regulated expression of RAD21. MiR-766-5p was identified functioning as a sponge for MIR4697HG and is sequestered by MIR4697HG. We also found either miR-766-5p inhibitor or PRR12 knockdown rescued the function depletion caused by MIR4697HG overexpression. In all, the down-regulated expression of MIR4697HG inhibited PRR12 to suppress glioma and led to the deterioration of glioma. CONCLUSION: RAD21-induced down-regulated expression of MIR4697HG is correlated with aggravation of glioma. The MIR4697HG/miR-766-5p/PRR12 axis predicts poor results in glioma and MIR4697HG could be considered as a promising biomarker for diagnosis and treatment of glioma.

Glibenclamide ameliorates the disrupted blood-brain barrier in experimental intracerebral hemorrhage by inhibiting the activation of NLRP3 inflammasome.

BACKGROUND: Glibenclamide is a widely used sulfonylurea drug prescribed to treat type II diabetes mellitus. Previous studies have demonstrated that glibenclamide has neuroprotective effects in central nervous system injury. However, the exact mechanism by which glibenclamide acts on the blood-brain barrier (BBB) after intracerebral hemorrhage (ICH) remains unclear. The purpose of this study was to validate the neuroprotective effects of glibenclamide on ICH and to explore the mechanisms underlying these effects. METHODS: We investigated the effects of glibenclamide on experimental ICH using the autologous blood infusion model. Glibenclamide was administrated either immediately or 2 hr after ICH. Brain edema was quantified using the wet-dry method 3 days after injury. BBB integrity was evaluated by Evans Blue extravasation and degradation of the tight junction protein zona occludens-1 (ZO-1). mRNA levels of inflammatory cytokines were determined by quantitative polymerase chain reaction. Activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome and cell viability were also measured in cerebral microvascular endothelial b.End3 cells exposed to hemin. Neurological changes were evaluated by the Garcia score and rotarod test. RESULTS: After ICH, the brain water content, Evans Blue extravasation, and inflammatory cytokines decreased significantly in the ipsilateral hemisphere of the experimental compared to the vehicle group. Glibenclamide treatment and NLRP3 knockdown significantly reduced hemin-induced activation of the NLRP3 inflammasome, release of extracellular lactate dehydrogenase, apoptosis, and loss of ZO-1 in b.End3 cells. However, NLRP3 knockdown abolished the protective effect of glibenclamide. CONCLUSION: Glibenclamide maintained BBB integrity in experimental ICH by inhibiting the activation of the NLRP3 inflammasome in microvessel endothelial cells. Our findings will contribute to elucidating the pharmacological mechanism of action of glibenclamide and to developing a novel therapy for clinical ICH.

Zinc-finger protein X-linked is a novel predictor of prognosis in patients with colorectal cancer.

Zinc-finger protein X-linked (ZFX) has been demonstrated to play an important role in the development of human malignancies. However, its prognostic significance in cancer patients remains unclear and less is known about its role in colorectal cancer (CRC). In this study, we found that the expression of ZFX in CRC tissues was significantly higher than that in corresponding normal tissues by quantitative real-time polymerase chain reaction and Western blot. Using immunohistochemistry, we explored the associations between protein expression of ZFX and clinicopathological parameters in 120 CRC cases. The results showed that ZFX expression was significantly associated with tumor differentiation (P = 0.022), tumor size (P = 0.037), tumor invasion (P = 0.027), lymph node metastasis (P = 0.042), distant metastasis (P = 0.011), and Dukes' classification (P = 0.028). Moreover, according to Kaplan-Meier model, patients with high expression of ZFX had a significantly poorer prognosis compared to those with low expression of ZFX. Multivariate analysis suggested that high expression of ZFX was an independent prognostic factor for CRC patients. In conclusion, our findings for the first time demonstrated that ZFX expression may be associated with the progress of CRC and suggested that ZFX has the potential value to be an effective prognostic predictor for CRC patients.

Impact of thoracic injury on traumatic brain injury outcome.

BACKGROUND: To assessed the significance of thoracic injury on the 30-day mortality and outcome of traumatic brain injury (TBI). METHODS: TBI patients admitted to our department were retrospectively evaluated. We developed two prognostic models based on admission predictors with logistic regression analysis to assess the significance of thoracic injuries in determining the 30-day mortality and outcome. The internal validity of the models was evaluated with the bootstrap re-sampling technique. We also validated the models in an external series of 165 patients that collected from our center. Discriminative ability was evaluated with C statistic. Calibrative ability was assessed with the Hosmer-Lemeshow test (H-L test). RESULTS: Among 505 TBI patients admitted, 102 (20.2%) had thoracic injuries. Patients with a PCS ≥ 6 had a 3.142 and 8.065 times higher odds of mortality and poor outcome compared with patients with a PCS <6, respectively. Any one-score increase of the TTS had a 1.193 times higher odds of a poor outcome (p = 0.017). The predictive model for mortality and 30-day functional outcome both had good accuracy (AUC: 0.875; 95% confidence interval [CI], 0.841-0.910 and AUC: 0.888; 95%CI, 0.860-0.916, respectively). Internal validation showed no over optimism in any of the two models' predictive C statistics (C statistic 0.872 for 30-day mortality and C statistic 0.884 for the 30-day neurological outcome). The external validation confirmed the discriminatory ability of these models (C statistic 0.949 (95%CI: 0.919-0.980) for 30-day mortality and C statistic 0.915 (95%CI: 0.868-0.963) for the 30-day neurological outcome). The calibration was also good for patients from the validation population (H-L test p>0.05). CONCLUSION: Thoracic injury diagnosed by CT has a negative impact on the 30-day mortality and functional outcome of TBI patients. The extent of PC and the TTS are the predictors for TBI outcome.

UHRF2 mRNA expression is low in malignant glioma but silencing inhibits the growth of U251 glioma cells in vitro.

UHRF2 is a member of the ubiquitin plant homeo domain RING finger family, which has been proven to be frequently up-regulated in colorectal cancer cells and play a role as an oncogene in breast cancer cells. However, the role of UHRF2 in glioma cells remains unclear. In this study, we performed real-time quantitative PCR on 32 pathologically confirmed glioma samples (grade I, 4 cases; grade II, 11 cases; grade III, 10 cases; and grade IV, 7 cases; according to the 2007 WHO classification system) and four glioma cell lines (A172, U251, U373, and U87). The expression of UHRF2 mRNA was significantly lower in the grade III and grade IV groups compared with the noncancerous brain tissue group, whereas its expression was high in A172, U251, and U373 glioma cell lines. An in vitro assay was performed to investigate the functions of UHRF2. Using a lentivirus-based RNA interference (RNAi) approach, we down-regulated UHRF2 expression in the U251 glioma cell line. This down- regulation led to the inhibition of cell proliferation, an increase in cell apoptosis, and a change of cell cycle distribution, in which S stage cells decreased and G2/M stage cells increased. Our results suggest that UHRF2 may be closely related to tumorigenesis and the development of gliomas.

A novel gene RNF138 expressed in human gliomas and its function in the glioma cell line U251.

BACKGROUND: The gliomas represent the most common primary malignant brain tumors; however, little is known about the molecular pathogenesis of these tumors. Recent research reveals that the oncogenesis and development of gliomas have a close relation to the overexpression of several oncogenes and the inactivation of tumor suppressor genes. Whether the RING finger protein, RNF138, a newly discovered protein, plays a role in glioma oncogenesis is unknown. The present study investigates the expression levels of RNF138 mRNA in glioma samples and noncancerous brain samples and its function in the human glioma cell line U251. METHODS: RT-PCR was used to ascertain the expression of RNF138 mRNA in the glioma cell lines U251, SHG44, U87, A172, and U373. The RNF138 mRNA expression levels of 35 pathological confirmed glioma samples (Grade I - 4 cases, Grade II - 13 cases, Grade III - 11 cases, and Grade IV - 7 cases) and five noncancerous brain tissue samples were analyzed by real-time quantitative PCR. By RNA interference (RNAi) with the lentivirus vector system, the expression of RNF138 was inhibited in the human astrocytomas-glioblastoma multiforme cell line U251. The effects of RNF138-knockdown on cell proliferation were assessed by Cellomics, and cell cycle and cell apoptosis were assessed by FACS. RESULTS: The RNF138 mRNA is expressed in the five glioma cell lines, and its expression level is significantly higher in glioma tissue than in noncancerous brain tissue. By down-regulation of RNF138 expression, U251 cell proliferation was inhibited and cell apoptosis increased. At the same time, S stage cells lessened and G2 stage cells increased. CONCLUSION: The RNF138 gene is highly expressed in glioma tissue and glioma cell lines. It plays an important role in glioma cell proliferation, apoptosis, and cell cycle.

The Zfx gene is expressed in human gliomas and is important in the proliferation and apoptosis of the human malignant glioma cell line U251.

BACKGROUND: Zfx is a zinc finger protein of the Zfy family, whose members are highly conserved in vertebrates. Zfx is a shared transcriptional regulator of both embryonic stem cells (ESC) and hematopoietic stem cells (HSC), which suggests a common genetic basis of self-renewal in embryonic and adult stem cells. The level of Zfx expression correlates with aggressiveness and severity in many cancer types, including prostate cancer, breast cancer, and leukemia. However, the importance of Zfx in human glioma is largely unknown. In the present study, we examined the role of Zfx in human glioma. METHODS: We detected expression levels of Zfx mRNA in U251 cells, U87 cells, U373 cells, and A172 cells by semi-quantitative RT-PCR. To analyze the expression of Zfx mRNA in glioma tissues, we performed real-time quantitative PCR on 35 pathologically confirmed glioma samples (Grade I-4cases, Grade II-13cases, Grade III-11cases, and Grade IV-7cases) and on 5 noncancerous brain tissue samples. We used lentivirus-mediated small interfering RNAs (siRNAs) to knock down Zfx expression in the human malignant glioma cell line U251. Changes in Zfx target gene expression were determined by real-time RT-PCR. Cell proliferation was examined by a High Content Screening assay. DNA synthesis in proliferating cells was determined by BrdU incorporation. Cell cycle distribution and apoptosis were detected by flowcytometric analysis. RESULTS: We discovered that Zfx mRNA was expressed in U251 cells, U87 cells, U373 cells, and A172 cells. The expression level of Zfx is significantly higher in gliomas compared to noncancerous brain tissue. Using a lentivirus-based RNAi approach, Zfx expression was significantly inhibited in human glioblastoma U251 cells. The effects of Zfx knockdown on cell proliferation, cell cycle distribution, and apoptosis were assessed. Inhibition of Zfx expression in U251 cells by RNAi significantly impaired cell proliferation, increased apoptosis, and arrested cells in S phase. CONCLUSIONS: The results of our study demonstrate that the Zfx gene is highly expressed in glioma tissue and in glioma cell lines. Furthermore, Zfx may play a critical role in cell proliferation, cell cycle distribution, and apoptosis of human malignant glioma cells.