RPN1 Is Associated With Immunosuppression in Pan-Cancer and Affects the Malignant Phenotype of Tumor.

Fructose 1,6-bisphosphatase 1 (FBP1) has been considered as a potential prognostic biomarker in glioblastoma (GBM), and this study explored the underlying mechanism. The expression and effect of FBP1 expression on the prognosis of GBM patients were examined applying bioinformatics analyses. After measuring the expression of FBP1 in normal glial cell line HEB and GBM cells, cell counting kit-8 (CCK-8), 5-ethynyl-2-deoxyuridine (EdU), colony formation, transwell, and wound healing assay were carried out to examine the effects of silencing FBP1 on the proliferation and invasion of GBM cells. Aerobic glycolysis was measured by calculating the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of FBP1-silenced GBM cells. Furthermore, the protein levels of the mediators related to PI3K/AKT pathway and BCL2 protein family were detected via immunoblotting. Additionally, the effects of FBP1 silencing on the macrophage M2 polarization were assessed based on the fluorescence intensity of CD206 and the phosphorylation of STAT6 quantified by immunofluorescence and immunoblotting, respectively. High-expressed FBP1 was indicative of a worse prognosis of GBM. FBP1 knockdown in GBM cells suppressed the proliferation, invasion, migration, and aerobic glycolysis of GBM cells, lowered the phosphorylation levels of AKT and PI3K and the protein expression of BCL2 but promoted BAX protein expression. Moreover, FBP1 knockdown reduced CD206 fluorescence intensity and the phosphorylation of STAT6. To conclude, FBP1 could be considered as a biomarker that affected the malignant phenotypes and aerobic glycolysis in GBM, contributing to the diagnosis and treatment of GBM.

The poor surgical efficacy and recurrence of glioblastoma (GBM) are due to its lack of visible infiltrative features. Our bioinformatics study suggests that low expression of small ubiquitin-like modifier (SUMO)-specific protease 7 (SENP7) indicates poor prognosis in GBM. This study investigated the effect of SENP7 expression on the invasion, migration, and proliferation of GBM cells and aims to identify the SUMO target proteins affected by SENP7. SENP7 expression was analyzed in eight GBM tumor samples and four GBM cell lines, comparing them to normal brain tissue. The effect of SENP7 overexpression on GBM LN229 cell migration, invasion, and proliferation was examined through in vitro assays. Furthermore, four SUMO target proteins involved in tumor invasion and proliferation (CDK6, matrix metalloproteinase-9 [MMP9], AKT, and HIF-1α) were studied to explore SENP7's molecular mechanism. SENP7 expression was significantly lower in GBM tumors compared to normal tissue. SENP7 overexpression in LN229 cells inhibited migration and invasion without affecting proliferation. Overexpression reduced the levels of MMP9, AKT, and HIF-1α, but not CDK6. Immunohistochemical analysis showed decreased MMP9 and CD31 levels, suggesting reduced tumor invasion and angiogenesis. However, SENP7 overexpression did not affect tumor growth in vivo. SENP7 inhibits GBM invasion by dissociating proteins associated with tumor invasion from SUMO2/3, providing a potential target for future GBM therapies.

EGFLAM as a novel gene biomarker has been reported in some cancers but not glioblastoma (GBM) yet. To clarify the functional role of EGFLAM in GBM, we performed this study. Firstly, based on TCGA and Oncomine database, EGFLAM expression and clinical significance in GBM patients was analyzed. Furthermore, the biological effect of EGFLAM in GBM cells was determined by qRT-PCR, CCK-8 assay, colony formation assay, wound healing assay, transwell assays and western blot analysis. The databases analysis showed that EGFLAM expression was at higher levels in GBM patients with poor prognosis. The results indicated that EGFLAM silence inhibited the proliferation, migration and invasion of U87 cells, which was regulated through repression of PI3K/AKT pathway. Accordingly, the data from our work shed some light on EGFLAM might be a prognostic biomarker and therapeutic target for GBM.

MicroRNAs (miRNAs) are acknowledged as essential regulators in human cancer types, including glioblastoma (GBM). However, the functions of microRNA-3666 (miR-3666) in GBM remain unclear. In the present study, it was identified that the expression of miR-3666 was significantly downregulated in GBM tissues compared with adjacent normal tissues by reverse transcription-quantitative polymerase chain reaction. Additionally, miR-3666 was downregulated in GBM cell lines. Furthermore, it was observed that the miR-3666 expression level in patients with GBM was associated with prognosis. With functional experiments, it was identified that overexpression of miR-3666 significantly inhibited the proliferation, migration and invasion of GBM cells in vitro by Cell Counting kit-8 and Transwell assays. Ectopic expression of miR-3666 significantly arrested GBM cells in the G0 phase by fluorescence activated cell sorting. In terms of the underlying mechanism, it was identified that lysine-specific demethylase 2A (KDM2A) is a direct target of miR-3666 in GBM cells. Overexpression of miR-3666 significantly decreased the expression of KDM2A in GBM cells. Furthermore, it was observed that knockdown of KDM2A significantly suppressed the proliferation, migration and invasion of GBM cells. Collectively, the present results demonstrated that the miR-3666/KDM2A axis serves an important role in the progression of GBM, which provides novel insight into the development of therapeutic strategies for GBM treatment.

Invasion and migration are the key hallmarks of cancer, and aggressive growth is a major factor contributing to treatment failure and poor prognosis in glioblastoma. Protein arginine methyltransferase 6 (PRMT6), as an epigenetic regulator, has been confirmed to promote the malignant proliferation of glioblastoma cells in previous studies. However, the effects of PRMT6 on glioblastoma cell invasion and migration and its underlying mechanisms remain elusive. Here, we report that PRMT6 functions as a driver element for tumor cell invasion and migration in glioblastoma. Bioinformatics analysis and glioma sample detection results demonstrated that PRMT6 is highly expressed in mesenchymal subtype or invasive gliomas, and is significantly negatively correlated with their prognosis. Inhibition of PRMT6 (using PRMT6 shRNA or inhibitor EPZ020411) reduces glioblastoma cell invasion and migration in vitro, whereas overexpression of PRMT6 produces opposite effects. Then, we identified that PRMT6 maintains the protein stability of EZH2 by inhibiting the degradation of EZH2 protein, thereby mediating the invasion and migration of glioblastoma cells. Further mechanistic investigations found that PRMT6 inhibits the transcription of TRAF6 by activating the histone methylation mark (H3R2me2a), and reducing the interaction between TRAF6 and EZH2 to enhance the protein stability of EZH2 in glioblastoma cells. Xenograft tumor assay and HE staining results showed that the expression of PRMT6 could promote the invasion of glioblastoma cells in vivo, the immunohistochemical staining results of mouse brain tissue tumor sections also confirmed the regulatory relationship between PRMT6, TRAF6, and EZH2. Our findings illustrate that PRMT6 suppresses TRAF6 transcription via H3R2me2a to enhance the protein stability of EZH2 to facilitate glioblastoma cell invasion and migration. Blocking the PRMT6-TRAF6-EZH2 axis is a promising strategy for inhibiting glioblastoma cell invasion and migration.

BackgroundOur previous bioinformatics-based study found that midkine (MDK) was associated with poor prognosis of glioblastoma (GBM). However, the mechanism of MDK in GBM remains elusive.MethodsA public GBM-related dataset and GBM tissues from our center were used validate the aberrant expression of MDK in GBM at the RNA and protein levels. The relationship between MDK expression and survival of GBM patients was also explored through survival analysis. Subsequently, we identified MDK-related GBM-specific genes using differential expression analysis. Functional enrichment analyses were performed to reveal their potential biological functions. CCK-8, 5-ethynyl-2′-deoxyuridine, and Matrigel-transwell assays were performed in GBM cell lines in which MDK was knocked out or overexpressed in order assess the effects of MDK on proliferation, migration, and invasion of GBM cells. Western blotting was performed to detect candidate proteins.ResultsOur study showed MDK is a promising diagnostic and prognostic biomarker for GBM because it is highly expressed in the disease and it is associated with poor prognosis. MDK is involved in various cancer-related pathways, such as PI3K-Akt signaling, the cell cycle, and VEGF signaling. A comprehensive transcriptional regulatory network was constructed to show the potential pathways through which MDK may be involved in GBM. In vitro, Overexpression of MDK augmented proliferation, migration, and invasion of GBM cell lines, whereas suppression of MDK led to the opposite effects. Furthermore, our study confirmed that MDK promotes the progression of GBM by activating the PI3K-Akt signaling pathway.ConclusionsOur present study proposes that MDK promotes GBM by activating the PI3K-Akt signaling pathway, and it describes a potential regulatory network involved.

BackgroundHomeobox (HOX) genes encode transcription factors that are critical to morphogenesis and cell differentiation. Although the dysregulation of several HOX genes in glioblastoma (GBM) has been reported, little is known about HOXC6 expression in GBM. Therefore, in this study, we investigated the expression levels of the HOXC6 in GBM and explored the regulatory mechanism underlying the role of HOXC6 in GBM progression.MethodsThe ONCOMINE and Oncolnc databases were used to predict the expression level of HOXC6 mRNA and its prognostic value in GBM. The expressions of HOXC6 mRNA in GBM tissues and adjacent brain tissues were detected using qRT-PCR and Western blot. Immunohistochemistry was performed to verify the HOXC6 protein expression in 107 GBM tissues. Kaplan–Meier and Cox analyses were performed to validate the correlation between HOXC6 expression and GBM prognosis. Lentivirus-mediated HOXC6 mRNA overexpression and interference system were established and transfected into U251 and U87 cell lines. CCK-8, colony formation, wound healing and transwell assay were utilized to evaluate the effects of HOXC6 on proliferation and migration of human GBM cells.ResultsHigh expression of HOXC6 was observed in GBM tissues and GBM cells lines, and it correlated with a decreased overall survival and disease-free survival. Overexpression of HOXC6 promoted the GBM cell proliferation and migration, whereas depletion of HOXC6 reduced GBM cell proliferation and migration. Mechanistic study showed that upregulation of HOXC6 significantly increased the phosphorylation of Jun amino-terminal kinase, ERK and P38, as well as the expression of mitogen-activated protein kinase (MAPK) signaling–related genes, including c-myc, c-jun and p53. Inversely, silencing HOXC6 showed the opposite results.ConclusionHOXC6 promoted proliferation and migration of GBM cells via the activation of MAPK pathway.

Background: Glioblastoma (GBM) is considered a clinically refractory malignant tumor due to its high recurrence and malignancy, invasiveness, and poor prognosis. The ethnomedicine Huafengdan (HFD) is prepared using several Chinese herbs by a complex fermentation process that has a long history. Previous studies have reported the inhibitory effect of HFD on GBM both in vitro and in vivo; however, its mechanism of action is unclear. Methods: The inhibitory effects of HFD on the growth, migration, and invasion of GBM cells were determined using the MTT assay, EdU assay, Transwell assay, flow cytometry, and Western blotting. A subcutaneous graft tumor model of nude BALB/c mice was established using U87 cells, and the in vivo activity and toxicity of HFD were evaluated using immunohistochemical staining and hematoxylin and eosin staining. Network pharmacology, bioinformatics, and transcriptomics were used to screen the targets and related signaling pathways of HFD in GBM and were validated using qPCR, CETSA, and Western blotting. Results: HFD inhibited the proliferation, invasion, and migration of GBM cells and induced S-phase block and apoptosis in GBM cells. It inhibited the in vivo growth of GBM cells without obvious toxicity. Mechanistic studies showed that the inhibition of GBM cell growth, migration, and invasion by HFD involved the key targets PLAU and CAV1. Its associated signaling pathways were the PI3K/Akt signaling pathway and cell cycle signaling pathway. Conclusions: Our findings confirm the novel function of HFD in inhibiting GBM cell growth in vitro and in vivo and highlight its potential in treating GBM.

BackgroundAs an adult tumor with the most invasion and the highest mortality rate, the inherent heterogeneity of glioblastoma (GBM) is the main factor that causes treatment failure. Therefore, it is important to have a deeper understanding of the pathology of GBM. Some studies have shown that Eukaryotic Initiation Factor 4A-3 (EIF4A3) can promote the growth of many people’s tumors, and the role of specific molecules in GBM remains unclear.MethodsThe correlation between the expression of EIF4A3 gene and its prognosis was studied in 94 GBM patients using survival analysis. Further in vitro and in vivo experiments, the effect of EIF4A3 on GBM cells proliferation, migration, and the mechanism of EIF4A3 on GBM was explored. In addition, combined with bioinformatics analysis, we further confirmed that EIF4A3 contributes to the progress of GBM.ResultsThe expression of EIF4A3 was upregulated in GBM tissues, and high expression of EIF4A3 is associated with poor prognosis in GBM. In vitro, knockdown of EIF4A3 significantly reduced the proliferation, migration, and invasion abilities of GBM cells, whereas overexpression of EIF4A3 led to the opposite effect. The analysis of differentially expressed genes related to EIF4A3 indicates that it is involved in many cancer-related pathways, such as Notch and JAK-STAT3 signal pathway. In Besides, we demonstrated the interaction between EIF4A3 and Notch1 by RNA immunoprecipitation. Finally, the biological function of EIF4A3-promoted GBM was confirmed in living organisms.ConclusionThe results of this study suggest that EIF4A3 may be a potential prognostic factor, and Notch1 participates in the proliferation and metastasis of GBM cells mediated by EIF4A3.

GNA13 inhibits glioblastoma metastasis via the ERKs/FOXO3 signaling pathway

PurposeWe previously reported that expression of dickkopf-3 (DKK3), which is involved in the Wnt/β-catenin pathway, is significantly associated with prognosis in patients with glioblastoma multiforme (GBM). The aim of this study was to compare the association of DKK3 with other Wnt/β-catenin pathway-related genes and immune responses between lower grade glioma (LGG) and GBM.MethodsWe obtained the clinicopathological data of 515 patients with LGG (World Health Organization [WHO] grade II and III glioma) and 525 patients with GBM from the Cancer Genome Atlas (TCGA) database. We performed Pearson’s correlation analysis to investigate the relationships between Wnt/β-catenin-related gene expression in LGG and GBM. Linear regression analysis was performed to identify the association between DKK3 expression and immune cell fractions in all grade II to IV gliomas.ResultsA total of 1,040 patients with WHO grade II to IV gliomas were included in the study. As the grade of glioma increased, DKK3 showed a tendency to be more strongly positively correlated with the expression of other Wnt/β-catenin pathway-related genes. DKK3 was not associated with immunosuppression in LGG but was associated with downregulation of immune responses in GBM. We hypothesized that the role of DKK3 in the Wnt/β-catenin pathway might be different between LGG and GBM.ConclusionAccording to our findings, DKK3 expression had a weak effect on LGG but a significant effect on immunosuppression and poor prognosis in GBM. Therefore, DKK3 expression seems to play different roles, through the Wnt/β-catenin pathway, between LGG and GBM.

Leukemia-Associated Rho Guanine Nucleotide Exchange Factor and Ras Homolog Family Member C Play a Role in Glioblastoma Cell Invasion and Resistance

Introduction Glioblastoma (GBM) is the most frequent and malignant type of primary brain tumors in adults. The valuable prognostic biomarkers and therapeutic targets for GBM remain to be elucidated. The association of adipokines with cancer has been well documented. The C1q/TNF-related protein 1 (CTRP1), a novel adipokine, belongs to the CTRP family. Methods In the present study, the expression and potential roles of CTRP1 in GBM were explored based on in silico evaluation, including GEPIA, the Pathology Atlas of the Human Protein Atlas, cBioPortal, TIMER, and SurvExpress. The CCK8, transwell, and wound healing assays were used to detect cell proliferation and migration. Results It was found that mRNA expression levels of CTRP1 were significantly upregulated in GBM tissues compared with those in nontumor tissues according to the analysis on public dataset and immunohistochemical results of GBM tissues (P<0.05). CTRP1 was mainly localized in the cytoplasm and cell membrane of GBM cells. The genetic alterations of CTRP1 occurred at a low rate in GBM (2 of 591 sequenced cases/patients, 0.33%). The mRNA expression levels of CTRP1 were positively associated with the tumor-infiltrating macrophages and CCL2 in GBM (P<0.05, respectively). The higher mRNA expression levels of CTRP1 were significantly correlated with higher risk and shorter overall survival time in GBM (P<0.05). CTRP1 knockdown significantly inhibited the proliferation and migration in human GBM cells, suggesting the inhibition of CTRP1 on human GMB progression. Moreover, CTRP1 knockdown inhibited CCL2 expression, and CCL2 overexpression reversed the inhibition of cell proliferation and migration induced by CTRP1 knockdown, suggesting that CTRP1 promoted tumor progression by regulating CCL2 expression. Conclusions These findings suggest that CTRP1 potentially indicates poor prognosis in GBM and promotes the progression of human GBM.

Glioblastoma (GBM) is the most universal and devastating primary intracranial neoplasm in the central nervous system. Urolithin A (UA) possesses many pharmacological and biological activities, but its function in GBM is not clear. CCK-8 and colony formation test were used to measure the anti-proliferative potency of UA against GBM cells. Flow cytometry was applied to evaluate cell cycle arrest and apoptosis of U251 and U118 MG cells upon UA incubation. Quantitative real-time PCR and western blotting were conducted to test the regulatory effect of UA on the expression of Sirt1 and FOXO1. Immunodeficient mice were implanted with GBM cells for in vivo validation of the anti-cancer effect of UA. We found UA repressed the proliferation, migration and invasion of glioblastoma cells, while also inhibiting the induction of colony formation ability and epithelial to mesenchymal transition (EMT) in a time- or dose-dependent manner. The does-dependent relationship of UA inducing the cell cycle arrest and apoptosis of glioblastoma cells was identified. Furthermore, UA could enhance the expression levels of Sirt1 and FOXO1 and the knockdown of Sirt1 blocked the inhibitory effects of UA on the proliferation and migration of glioblastoma cells and correspondingly modified the expression level of FOXO1. Overexpression of Sirt1 restored the despaired inhibitory effect of UA induced by Sirt1 knockout on the proliferation and migration of glioblastoma cells. In animal experiments, UA decreased the tumor size and weight of glioblastoma in xenograft nude mice and promoted the expression of Sirt1 and FOXO1 in transplanted tumors. Our findings presented in this study indicate that UA exerts a repressive effect on glioblastoma cells in vivo and in vitro by regulating the Sirt1-FOXO1 axis via the ERK and AKT pathways, indicating that UA is a new novel therapeutic candidate for the treatment of glioblastoma.

Long noncoding RNAs (lncRNAs) have been acknowledged as important regulators in various human cancers. lncRNA MNX1-AS1 has been shown to be an oncogene in epithelial ovarian cancer. However, the function of MNX1-AS1 in glioblastoma (GBM) remains largely unknown. Here we found that the expression of MNX1-AS1 was significantly upregulated in GBM tissues and cell lines. Knockdown of MNX1-AS1 significantly inhibited the proliferation, migration, and invasion of GBM cells. In terms of mechanism, we found that MNX1-AS1 could bind to miR-4443 in GBM cells. Overexpression of miR-4443 significantly inhibited the expression of MNX1-AS1 and vice versa. Moreover, there was an inverse correlation between the expression levels of MNX1-AS1 and miR-4443 in GBM tissues. We found that overexpression of miR-4443 inhibited the proliferation, migration, and invasion of GBM cells. We also showed that inhibition of miR-4443 reversed the effects of MNX1-AS1 knockdown on GBM cell proliferation, migration, and invasion. Taken together, we found that MNX1-AS1 promoted the proliferation, migration, and invasion of GBM cells through inhibiting miR-4443.