CRISPR screening of E3 ubiquitin ligases reveals Ring Finger Protein 185 as a novel tumor suppressor in glioblastoma repressed by promoter hypermethylation and miR-587.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor. E3 ligases play important functions in glioma pathogenesis. CRISPR system offers a powerful platform for genome manipulation, while the screen of E3 ligases in GBM still remains to be explored. Here, we first constructed an E3 ligase small guide RNA (sgRNAs) library for glioma cells growth screening. After four passages, 299 significantly enriched or lost genes (SELGs) were compared with the initial state. Then the clinical significance of SELGs were validated and analyzed with TCGA glioblastoma and CGGA datasets. As RNF185 showed lost signal, decreased expression and favorable prognostic significance, we chose RNF185 for functional analysis. In vitro overexpressed cellular phenotype showed that RNF185 was a tumor suppressor in two glioma cell lines. Finally, the molecular mechanism of decreased RNF185 expression was investigated and increased miR-587 expression and DNA hypermethylation was evaluated. This study would provide a link between the molecular basis and glioblastoma pathogenesis, and a novel perspective for glioblastoma treatment.

Gamma-glutamylcyclotransferase promotes the growth of human glioma cells by activating Notch-Akt signaling.

Glioma as an aggressive type tumor is rapidly growing and has become one of the leading cause of cancer-related death worldwide. γ-Glutamylcyclotransferase (GGCT) has been shown as a diagnostic marker in various cancers. To reveal whether there is a correlation between GGCT and human glioma, GGCT expression in human glioma tissues and cell lines was first determined. We found that GGCT expression was up-regulated in human glioma tissues and cell lines. Further, we demonstrate that GGCT knockdown inhibits glioma cell T98G and U251 proliferation and colony formation, whereas GGCT overexpression leads to oppose effects. GGCT overexpression promotes the expression of Notch receptors and activates Akt signaling in glioma cells, and Notch-Akt signaling is activated in glioma tissues with high expression of GGCT. Finally, we show that inhibition of Notch-Akt signaling with Notch inhibitor MK-0752 blocks the effects of GGCT on glioma proliferation and colony formation. In conclusion, GGCT plays a critical role in glioma cell proliferation and may be a potential cancer therapeutic target.

Single-Cell Transcriptome Reveals Cell Type-Specific Molecular Pathology in a 2VO Cerebral Ischemic Mouse Model.

Post-ischemia memory impairment is a major sequela in cerebral ischemia patients. However, cell type-specific molecular pathology in the hippocampus after ischemia is poorly understood. In this study, we adopted a mouse two-vessel occlusion ischemia model (2VO model) to mimic cerebral ischemia-induced memory impairment and investigated the single-cell transcriptome in the hippocampi in 2VO mice. A total of 27,069 cells were corresponding 14 cell types with neuronal, glial, and vascular lineages. We next analyzed cell-specific gene alterations in 2VO mice and the function of these cell-specific genes. Differential expression analysis identified cell type-specific genes with altered expression in neurons, astrocytes, microglia, and oligodendrocytes in 2VO mice. Notably, four subtypes of oligodendrocyte precursor cells with distinct differentiation pathways were suggested. Taken together, this is the first single-cell transcriptome analysis of gene expression in a 2VO model. Furthermore, we suggested new types of oligodendrocyte precursor cells with angiogenesis and neuroprotective potential, which might offer opportunities to identify new avenues of research and novel targets for ischemia treatment.

The GGCT and REST positive feedback loop promotes tumor growth in Glioma.

BACKGROUND: γ-Glutamylcyclotransferase (GGCT), an enzyme crucial in glutathione metabolism, has emerged as a participant in tumorigenesis. The present study is designed to elucidate the biological role and molecular mechanisms underlying GGCT in glioma. METHODS: Gene Expression Profiling Interactive Analysis (GEPIA), Chinese Glioma Genome Atlas (CGGA), and PrognoScan online databases were utilized to examine the expressions and clinical prognosis of GGCT and REST in glioma. Cell Counting Kit-8 (CCK-8), Transwell, Wound healing, and Flow cytometric assays, and RNA-sequencing analysis were employed to uncover the molecular role of GGCT and REST. Prediction of Differentially expressed microRNA (DE-miRNAs) and miRNAs targeting GGCT 3' Untranslated Region (UTR) was performed using miRanda online datasets. Finally, Real time-quantitative Polymerase Chain Reaction (RT-qPCR), western blot and dual luciferase reporter gene activity analysis were employed to confirm a positive feedback loop involving GGCT/REST/miR-34a-5p in glioma cells. RESULTS: High expression of GGCT was correlated with poor prognosis in glioma. GGCT silencing demonstrated inhibitory effects on the proliferation, migration, and induction of apoptosis in T98G and U251 cells. Mechanistically, GGCT downregulated REST expression and modulated cancer-associated pathways in glioma cells. High expression of REST was associated with poor prognosis in glioma. In vitro and in vivo experiments showed that REST overexpression restored the repression of proliferation, invasion, migration, and xenograft tumor formation induced by GGCT knockdown. Furthermore, the study uncovered that REST inhibited miR-34a-5p mRNA expression, and miR-34a-5p suppressed GGCT expression by targeting its 3'UTR, forming a positive regulatory loop in glioma. Notably, the inhibitor of miR-34a-5p restored the role of REST silencing in decreasing GGCT expression in glioma cells. CONCLUSIONS: GGCT/REST/miR-34a-5p axis holds promising potential as a therapeutic target, offering a potential breakthrough in the treatment of glioma.

Bromodomain-containing protein 4 activates cardiotrophin-like cytokine factor 1, an unfavorable prognostic biomarker, and promotes glioblastoma in vitro.

Background: Glioblastoma (GBM) is one of the most common and malignant brain tumors. Cardiotrophin-like cytokine factor 1 (CLCF1) is a member of the IL-6 superfamily. However, the clinical significance, potential role, and molecular mechanism of CLCF1 in GBM remain obscure. Here, the expression and prognostic significance of CLCF1 was investigated in GBM. Methods: The Cancer Genome Atlas (TCGA) GBM and Chinese Glioma Genome Atlas (CGGA) datasets were downloaded and analyzed by using Gene Expression Profiling Interactive Analysis (GEPIA). Next, 3 shRNAs targeting CLCF1 were designed, and silencing efficiency was examined with real-time polymerase chain reaction (PCR). Cell Counting Kit 8 (CCK-8), flow cytometry, transwell, and wound healing assays were used to study the function of CLCF1 in glioma cells. Results: We found increased expression of CLCF1 as an unfavorable prognostic marker in GBM. Functionally, down-regulation of CLCF1 significantly reduced cell proliferation, induced cell apoptosis and cell cycle G2 phase arrest, and weakened the migration and invasion of GBM cells. Downstream pathway analysis was conducted, and potential targets in cytokine receptors, extracellular matrix (ECM) receptors, apoptosis, and the cell cycle were uncovered. Finally, transcriptional regulators were analyzed, and bromodomain-containing protein 4 (BRD4) was found to activate CLCF1 in GBM. Conclusions: CLCF1, transcriptionally activated by BRD4, promotes glioma and serves as an unfavorable marker in GBM.

Immune-related lncRNAs, LINC01268 and CTB-31O20.2, as favorable prognostic markers for glioma inhibition.

Background: Glioblastoma (GBM) is the most common and fatal tumor in the central nervous system. Recent studies have found that long non-coding RNAs (lncRNAs) serve as competitive endogenous RNAs (ceRNAs) and play an important role in GBM by regulating immune responses. The aim of the present study was to identify lncRNAs with immune relevance and functions in GBM. Methods: We analyzed GBM datasets from The Cancer Genome Atlas (TCGA) database to obtain 356 significantly differentially expressed lncRNAs (DE-lncRNAs), 4,951 DE-mRNAs, and 34 DE-miRNAs in GBM, respectively. For mRNAs, 369 DE-mRNAs were identified as immune-related genes in the ImmPort database. For DE-lncRNAs, univariate analysis identified 39 DE-lncRNAs with prognostic significance, and 9 DE-lncRNAs were included in the ImmLnc database. Combined analysis was then conducted by integrating 9 immune-related DE-lncRNAs, 369 immune-related DE-mRNAs, and 34 DE-miRNAs. A ceRNA network composed of 2 upregulated lncRNAs (LINC01268 and CTB-31O20.2), 3 downregulated miRNAs, and 5 upregulated mRNAs was generated. Results: Kaplan-Meier survival analysis and univariate and multivariate Cox regression analyses showed that LINC01268 and CTB-31O20.2 serve as independent favorable prognostic markers in GBM. LINC01268 and CTB-31O20.2 overexpression was conducted in GBM cell U251. Cell Counting Kit-8 (CCK8), Transwell assay, and scratch healing assay indicated that LINC01268 and CTB-31O20.2 inhibit GBM cell line, U251, proliferation, invasion, and migration. Conclusions: LINC01268 and CTB-31O20.2 are independent prognostic immune-related markers, and reduce cancer cell proliferation and metastasis in GBM.

Bromodomain containing 4 transcriptionally activated Deltex E3 ubiquitin ligase 2 contributes to glioma progression and predicts an unfavorable prognosis.

Background: Glioblastoma multiforme (GBM) is the most common type of glioma, and the most aggressive brain malignancy in adults. This study sought to identify novel survival-status related markers, and examine their function in glioma. Methods: The gene expression, survival heatmaps, and Kaplan-Meier survival plots of the genes were analyzed by using gene expression profiling interactive analysis (GEPIA) dataset, Linked Omics. The single-cell data analysis and tumor immune infiltration analysis was conducted by Tumor Immune Estimation Resource (TIMER) dataset. DBTRG and U251 cells with silenced Deltex E3 ubiquitin ligase 2 (DTX2) expression were constructed and used for Cell Counting Kit 8 (CCK-8), and wound healing assay in vitro. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis was used to explore the histone activation marks and transcription factors DTX2 promoter. Dual-luciferase assays were carried out to detect the luciferase activities of bromodomain containing 4 (BRD4) binding to DTX2. Results: We first conducted a survival-status analysis to identify survival status-related genes in The Cancer Genome Atlas GBM and low-grade glioma data sets. A subsequent analysis identified 3 novel prognostic biomarkers; that is, DTX2, cytochrome P450 oxidoreductase, and Williams-Beuren syndrome chromosomal region 16 protein. In the validation Chinese Glioma Genome Atlas data sets, DTX2 showed the best performance, and was examined in a further analysis. Next, 3 short-hairpin ribonucleic acids were designed to silence DTX2 expression, and CCK-8 and wound-healing assays were applied to study the function of DTX2. We found that DTX2-silenced glioma cells exhibited a significant decrease in their growth and migration capabilities. Finally, the molecular basis for increased DTX2 in glioma was investigated via ChIP-Seq analysis and luciferase assays. The analysis revealed that DTX2 was transcriptionally activated by BRD4. Conclusions: In conclusion, BRD4 transcriptionally activates DTX2, contributes to glioma progression, predicts an unfavorable prognosis, and could provide new options for glioma prognosis prediction and treatment.

Hypomethylation-induced prognostic marker zinc finger DHHC-type palmitoyltransferase 12 contributes to glioblastoma progression.

Background: Glioma is the most common intracranial primary malignancy, characterized by abnormal signal transductions caused by transcriptional and post-transcriptional regulators. Studies show the palmitoylation of oncoproteins and tumor suppressors participate in cancer progression, while studies of protein S-palmitoyltransferases in glioma are limited. A systematic analysis of zinc finger DHHC-type palmitoyltransferases (ZDHHC) in glioma is still lacking. Methods: A prognostic heatmap and Kaplan-Meier overall survival plot of 24 members of the ZDHHC family in pan-cancer created. The expression and prognostic significance of ZDHHC12 was analyzed by using Gene Expression Profiling Interactive Analysis (GEPIA) and PrognoScan. DBTRG and U251 cells with silenced ZDHHC12 expression were constructed and used for cell counting kit-8 (CCK-8), Transwell assay and wound healing assay in vitro. Results: Here, we first conducted expression and prognostic analyses of 24 ZDHHCs from The Cancer Genome Atlas (TCGA), the Chinese Glioma Genome Atlas (CGGA), and other glioma datasets. We found ZDHHC12 to be the only unfavorable prognostic marker in glioma. The function of ZDHHC12 in glioma was then investigated with loss-of-function strategies and in vitro cell assays. Results showed that ZDHHC12 knockdown remarkably reduced the growth, migration, and invasion capabilities in DBTRG and U251 cell lines, suggesting that ZDHHC12 may contribute to malignant behavior in glioma cells. Finally, the molecular basis for ZDHHC12 expression in glioma was analyzed, and DNA hypomethylation was found to be responsible for increased ZDHHC12 mRNA expression and related prognoses. Conclusions: ZDHHC12 positively promoted the proliferation and migration of glioma cells. Decreased DNA methylation may lead to increased ZDHHC12 expression in gliomas. This study may deepen the understanding of glioma progression and therapeutics.

Decreased pygopus 2 expression suppresses glioblastoma U251 cell growth.

Gliomas are common malignant tumors of the human neural system, and Wnt signaling activation is closely related to glioma malignancy. Human Pygopus 2 (Pygo2) was recently discovered to be a component of the Wnt signaling pathway, which is required for ß-catenin/Tcf-dependent transcription. However, the role of Pygo2 in glioblastoma cell growth and survival remains uncertain. In the present study, Pygo2 expression was evaluated in 80 glioma tissue samples. Results demonstrated that tumor grade exhibited a positive correlation with overexpression of Pygo2. In addition, small hairpin RNA (shRNA) was used to specifically knockdown Pygo2 expression in human glioblastoma U251 cell lines. Results showed that inhibition of Pygo2 expression resulted in inhibited cell proliferation and invasiveness, as well as increased cell cycle arrest at the G(1) stage and decreased expression of the Wnt target gene cyclin D1. These results demonstrated that Pygo2 was highly expressed in glioma tissue and required for growth of glioblastoma cells.

Inhibition of Human Glioma Cell Proliferation Caused by Knockdown of Utrophin Using a Lentivirus-Mediated System.

BACKGROUND: Glioma is the most devastating brain tumor worldwide. Previous studies showed that UTRN (utrophin) was related to cancers, but its role in glioma cells remains uncovered. MATERIALS AND METHODS: RNAi was used to knockdown UTRN in U251 cells using lentivirus system. The knockdown efficiency was validated by real-time quantitative PCR. Cell proliferation, cell cycle, and apoptosis progression were determined by MTT, colony formation analysis, and flow cytometry analysis. Furthermore, some apoptotic markers were examined by Western blot assay. RESULTS: Most cells were infected. Cell proliferation and colony formation ability were suppressed in U251 cells lacking UTRN. Moreover, there was an obvious increase in cell percentage in the G2/M phases and a significant apoptosis in U251 cells after UTRN silencing. Further investigation demonstrated that UTRN knockdown activated caspase and PARP pathways. CONCLUSIONS: Knockdown of UTRN expression by shRNA evidently inhibited cell proliferation and promoted cell apoptosis in glioma cells.

Dural Tail Sign in the Resection of Ventral Foramen Magnum Meningiomas via a Far Lateral Approach: Surgical Implications.

OBJECTIVE: To investigate the implications of dural tail sign (DTS) in the tailored far lateral approach for resection of ventral foramen magnum meningiomas (FMMs). METHODS: Clinical data for 16 patients treated surgically for ventral FMMs over 5 years were reviewed retrospectively. RESULTS: The DTS was positive in 11 cases (68.8%) and negative in 5 cases (31.2%). The most frequent form was a single cranial tail (7 of 11), followed by multiple tails consisting of a cranial tail and a caudal tail (3 of 11), and multiple tails composed of a cranial tail and a contralateral tail (1 of 11). The retrocondylar approach was carried out in 5 cases without DTS characterized by a narrow dural attachment and a partial transcondylar approach in 11 cases with DTS featuring a broad and hypervascular dural attachment. Drilling ranged from approximately one fifth to one third of the condyle with reference to the DTS form and tumor size. Total tumor removal was achieved in 16 patients. Postoperative complications were encountered in 25% of patients, predominantly associated with cranial nerve impairment. Follow-up ranging from 8 to 56 months (mean 24.4 months) showed no tumor recurrence. CONCLUSIONS: In addition to tumor dural attachment and tumor size, we propose that DTS should be considered as another factor in planning the surgical approach for ventral FMMs. Differentiation between a positive and negative DTS plays a role in the neurosurgical planning of ventral FMMs. Bone removal is warranted in tumors with DTS, particularly the multiple form with contralateral tails, to facilitate the surgical procedure and achieve a more radical resection.

Huge familial colloid cyst of the third ventricle: An extraordinary presentation.

BACKGROUND: Since the use of computed tomography and magnetic resonance imaging, colloid cysts (CCs) are discovered more frequently and subsequently their true incidence exceeds the numbers previously estimated. In 1986, the first familial case was reported in two identical twin brothers. To date, a total of 17 of these cases have been reported, all differing in the pattern of affected family members. CASE DESCRIPTION: Here, we describe a unique presentation of a familial case and review the relevant literature on CCs and their natural history to improve our understanding of these cases. CONCLUSION: Familial CC can present in various patterns, sizes, and forms. A genetic factor is likely to be responsible in these cases, and further research is warranted to clarify this phenomenon.

Reduction cranioplasty with the aid of simulated computer imaging for the treatment of hydrocephalic macrocephaly.

OBJECT: The occurrence of hydrocephalic macrocephaly is uncommon. When the condition does occur, it is usually seen in infants and young children. Patients with this disorder have an excessively enlarged head and weak physical conditions. Various surgical techniques of reduction cranioplasty for the treatment of these patients have been reported. In this study, a revised surgical procedure with the aid of simulated computer imaging for the treatment of hydrocephalic macrocephaly is presented. METHODS: Five cases of hydrocephalic macrocephaly in children ranging in age from 16 to 97 months were reviewed. These patients underwent surgical treatment at The First Affiliated Hospital of Xiamen University over a period of 4 years from January 2007 to January 2011. After physical examination, a 3D computer imaging system to simulate the patient's postoperative head appearance and bone reconstruction was established. Afterward, for each case an appropriate surgical plan was designed to select the best remodeling method and cranial shape. Then, prior to performing reduction remodeling surgery in the patient according to the computer-simulated procedures, the surgeon practiced the bone reconstruction technique on a plaster head model made in proportion to the patient's head. In addition, a sagittal bandeau was used to achieve stability and bilateral symmetry of the remodeled cranial vault. Each patient underwent follow-up for 6-32 months. RESULTS: Medium-pressure ventriculoperitoneal shunt surgery or shunt revision procedures were performed in each patient for treating hydrocephalus, and all patients underwent total cranial vault remodeling to reduce the cranial cavity space. Three of the 5 patients underwent a single-stage surgery, while the other 2 patients underwent total cranial vault remodeling in the first stage and the ventriculoperitoneal shunt operation 2 weeks later because of unrecovered hydrocephalus. All patients had good outcome with regard to hydrocephalus and macrocephaly. CONCLUSIONS: There are still no standard surgical strategies for the treatment of hydrocephalic macrocephaly. Based on their experience, the authors suggest using a computer imaging system to simulate a patient's postoperative head appearance and bone reconstruction together with total cranial vault remodeling with shunt surgery in a single-stage or 2-stage procedure for the successful treatment of hydrocephalic macrocephaly.

The role of Pygopus 2 in rat glioma cell growth.

Glioma is a common malignant tumor of the human neural system, and Wnt signaling activation is closely connected with glioma malignancy. Pygopus 2 (Pygo2) was recently discovered as a component of the Wnt signaling pathway regulating ß-catenin/Tcf dependent transcription. However, the role of Pygo2 in glioma cells has not yet been defined. In the current study, we investigated the role of Pygo2 in rat glioma C6 cells for the first time. Our results showed that over-expression of Pygo2 promoted cell proliferation as well as enhanced cell cycle progression from G1 to S phase associated with an increase in the expression of the Wnt target gene cyclin D1. In contrast, knockdown of Pygo2 suppressed cell proliferation with cell cycle block from G1 to S phase and down-regulation of cyclin D1. In addition, the expression of Pygo2 and cyclin D1 in 67 glioma tissue samples was quantified by real-time reverse transcription polymerase chain reaction (RT-PCR) and immunochemistry. The data indicated that tumor grade was significantly associated with over-expression of Pygo2 and cyclin D1. We conclude that Pygo2 is highly expressed in and promotes the growth of glioma cells by an increase in the expression of cyclin D1 to improve G1/S transition.