MicroRNA-588 regulates the invasive, migratory and vasculogenic mimicry-forming abilities of hypoxic glioma cells by targeting ROBO1.

BACKGROUND: The microenvironment of hypoxia is an important factor contributing to the development of glioblastoma (GBM). MicroRNA-588 and its potential target Roundabout-directed receptor 1 (ROBO1) have been reported to promote tumor invasion and proliferation in diseases such as gastric, pancreatic and hepatocellular carcinoma, while their function in GBM and response to hypoxic states remain elusive. METHODS: A microarray was leveraged to identify differentially expressed microRNAs in U251 glioma cells cultured under normoxic and hypoxic conditions. The expression of miR-588 was assessed using quantitative real-time PCR (qRT‒PCR). Gain- and loss-of-function studies were used to evaluate the role of miR-588 under hypoxic and normoxic conditions. Cell invasion, migration, proliferation, and vasculogenic mimicry (VM) formation experiments were performed. The relationship between miR-588 and ROBO1 was confirmed using western blot and luciferase reporter assays. Intracranial xenograft tumor mouse models were used to study the function of miR-588 in vivo. RESULTS: The expression of miR-588 was significantly upregulated in hypoxic glioma cells relative to normoxic glioma cells. miR-588 inhibited the invasive, migratory and VM-forming abilities of glioma cells in vitro and in vivo. Mechanistically, roundabout guidance receptor 1 (ROBO1) is a direct, functionally relevant target of miR-588 in glioma. ROBO1 knockdown suppressed the expression of matrix metallopeptidase 2 (MMP2) and matrix metallopeptidase 9 (MMP9), thereby inhibiting the invasive, migratory and VM-forming abilities of glioma. CONCLUSIONS: MiR-588 regulated the behaviors of hypoxic glioma cells by targeting ROBO1. miR-588 can be used as a prognostic marker for glioma and has potential implications in glioma gene therapy.

RGS16 regulated by let-7c-5p promotes glioma progression by activating PI3K-AKT pathway.

Gliomas are the most common central nervous system tumours; they are highly aggressive and have a poor prognosis. RGS16 belongs to the regulator of G-protein signalling (RGS) protein family, which plays an important role in promoting various cancers, such as breast cancer, pancreatic cancer, and colorectal cancer. Moreover, previous studies confirmed that let-7c-5p, a well-known microRNA, can act as a tumour suppressor to regulate the progression of various tumours by inhibiting the expression of its target genes. However, whether RGS16 can promote the progression of glioma and whether it is regulated by miR let-7c-5p are still unknown. Here, we confirmed that RGS16 is upregulated in glioma tissues and that high expression of RGS16 is associated with poor survival. Ectopic deletion of RGS16 significantly suppressed glioma cell proliferation and migration both in vitro and in vivo. Moreover, RGS16 was validated as a direct target gene of miR let-7c-5p. The overexpression of miR let-7c-5p obviously downregulated the expression of RGS16, and knocking down miR let-7c-5p had the opposite effect. Thus, we suggest that the suppression of RGS16 by miR let-7c-5p can promote glioma progression and may serve as a potential prognostic biomarker and therapeutic target in glioma.

Cell senescence-associated genes predict the malignant characteristics of glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most malignant, aggressive and recurrent primary brain tumor. Cell senescence can cause irreversible cessation of cell division in normally proliferating cells. According to studies, senescence is a primary anti-tumor mechanism that may be seen in a variety of tumor types. It halts the growth and spread of tumors. Tumor suppressive functions held by cellular senescence provide new directions and pathways to promote cancer therapy. METHODS: We comprehensively analyzed the cell senescence-associated genes expression patterns. The potential molecular subtypes were acquired based on unsupervised cluster analysis. The tumor immune microenvironment (TME) variations, immune cell infiltration, and stemness index between 3 subtypes were analyzed. To identify genes linked with GBM prognosis and build a risk score model, we used weighted gene co-expression network analysis (WGCNA), univariate Cox regression, Least absolute shrinkage and selection operator regression (LASSO), and multivariate Cox regression analysis. And the correlation between risk scores and clinical traits, TME, GBM subtypes, as well as immunotherapy responses were estimated. Immunohistochemistry (IHC) and cellular experiments were performed to evaluate the expression and function of representative genes. Then the 2 risk scoring models were constructed based on the same method of calculation whose samples were acquired from the CGGA dataset and TCGA datasets to verify the rationality and the reliability of the risk scoring model. Finally, we conducted a pan-cancer analysis of the risk score, assessed drug sensitivity based on risk scores, and analyzed the pathways of sensitive drug action. RESULTS: The 3 potential molecular subtypes were acquired based on cell senescence-associated genes expression. The Log-rank test showed the difference in GBM patient survival between 3 potential molecular subtypes (P = 0.0027). Then, 11 cell senescence-associated genes were obtained to construct a risk-scoring model, which was systematically randomized to distinguish the train set (n = 293) and the test set (n = 292). The Kaplan-Meier (K-M) analyses indicated that the high-risk score in the train set (P < 0.0001), as well as the test set (P = 0.0053), corresponded with poorer survival. In addition, the high-risk score group showed a poor response to immunotherapy. The reliability and credibility of the risk scoring model were confirmed according to the CGGA dataset, TCGA datasets, and Pan-cancer analysis. According to drug sensitivity analysis, it was discovered that LJI308, a potent selective inhibitor of RSK pathways, has the highest drug sensitivity. Moreover, the GBM patients with higher risk scores may potentially be more beneficial from drugs that target cell cycle, mitosis, microtubule, DNA replication and apoptosis regulation signaling. CONCLUSION: We identified potential associations between clinical characteristics, TME, stemness, subtypes, and immunotherapy, and we clarified the therapeutic usefulness of cell senescence-associated genes.

Identification and validation of an anoikis-associated gene signature to predict clinical character, stemness, IDH mutation, and immune filtration in glioblastoma.

Background: Glioblastoma (GBM) is the most prominent and aggressive primary brain tumor in adults. Anoikis is a specific form of programmed cell death that plays a key role in tumor invasion and metastasis. The presence of anti-anoikis factors is associated with tumor aggressiveness and drug resistance. Methods: The non-negative matrix factorization algorithm was used for effective dimension reduction for integrated datasets. Differences in the tumor microenvironment (TME), stemness indices, and clinical characteristics between the two clusters were analyzed. Difference analysis, weighted gene coexpression network analysis (WGCNA), univariate Cox regression, and least absolute shrinkage and selection operator regression were leveraged to screen prognosis-related genes and construct a risk score model. Immunohistochemistry was performed to evaluate the expression of representative genes in clinical specimens. The relationship between the risk score and the TME, stemness, clinical traits, and immunotherapy response was assessed in GBM and pancancer. Results: Two definite clusters were identified on the basis of anoikis-related gene expression. Patients with GBM assigned to C1 were characterized by shortened overall survival, higher suppressive immune infiltration levels, and lower stemness indices. We further constructed a risk scoring model to quantify the regulatory patterns of anoikis-related genes. The higher risk score group was characterized by a poor prognosis, the infiltration of suppressive immune cells and a differentiated phenotype, whereas the lower risk score group exhibited the opposite effects. In addition, patients in the lower risk score group exhibited a higher frequency of isocitrate dehydrogenase (IDH) mutations and a more sensitive response to immunotherapy. Drug sensitivity analysis was performed, revealing that the higher risk group may benefit more from drugs targeting the PI3K/mTOR signaling pathway. Conclusion: We revealed potential relationships between anoikis-related genes and clinical features, TME, stemness, IDH mutation, and immunotherapy and elucidated their therapeutic value.

MiR-449a Sponges Cyclin D1 Expression and Suppresses Phosphorylation of Retinoblastoma Protein against Osteosarcoma.

MiR-449a has tumor-regulatory properties in different cancers, but its role in osteosarcoma had not been clearly understood. This research aimed to study the underlying mechanism of how miR-449a regulates osteosarcoma cells. The expression of miR-449a, cyclin D1, and pRb in osteosarcoma tissues and cultivated cells was assessed by qRT-PCR, western blot, and immunofluorescent assay. Dual-luciferase reporter assay was conducted to verify the target of miR-449a. Western blot, CCK-8, colony formation, and flow cytometry assays were applied to examine the regulatory effects of miR-449a on osteosarcoma cells and the molecular mechanism. MiR-449a and pRb expression was impeded whereas cyclin D1 expression was enhanced in osteosarcoma tissues and cells. Cyclin D1 was confirmed as a target of miR-449a. MiR-449a impeded the viability and proliferation of osteosarcoma cells and controlled the cell cycle through targeting cyclin D1 to reduce pRB phosphorylation. These findings provided evidence that miR-449a played an anticancer role in osteosarcoma cells by directly targeting cyclin D1 to prevent pRb from phosphorylation. Our study suggested that miR-449a might have the potential to become a new therapeutic biomarker involved in the diagnosis, prevention, and treatment of osteosarcoma.

Cullin-7 (CUL7) is overexpressed in glioma cells and promotes tumorigenesis via NF-κB activation.

BACKGROUND: Cullin-7 (CUL7) is a member of the DOC domain-containing cullin family and is involved in the regulation of cell transformation. However, the clinical significance, potential mechanism and upstream regulators of CUL7 in malignant gliomas remain to be determined. METHODS: Expression level data and clinical information were obtained via the Cancer Genome Atlas (TCGA) database, the Chinese Glioma Genome Atlas (CGGA) database, immunohistochemistry (IHC) and western blot analysis. Gene set enrichment analysis (GSEA) was used to explore the potential molecular mechanisms of CUL7. RNA silencing was performed using siRNA or lentiviral constructs in U87MG and U251 glioma cell lines and GSC267 glioma stem cells. CUL7 overexpression was performed using the GV141-CUL7 plasmid construct. In addition, overexpression of miR-3940-5p was performed and validated by quantitative real-time PCR (qRT-PCR). Cells were characterized in vitro or in vivo to evaluate their molecular status, cell proliferation, invasion, and migration by Cell Counting Kit (CCK)-8, EdU, flow cytometry, colony formation, Transwell and 3D tumour spheroid invasion assays. Coimmunoprecipitation (co-IP) and western blotting were performed to test the mechanisms of activation of the NF-κB signalling pathway. RESULTS: High CUL7 expression was associated with a high tumour grade, a mesenchymal molecular glioma subtype and a poor prognosis in patients. Gene silencing of CUL7 in U87MG and U251 cells significantly inhibited tumour growth, invasion and migration in vitro and in vivo. Western blot analysis revealed that cyclin-dependent kinase inhibitors and epithelial-mesenchymal transition (EMT) molecular markers changed under CUL7 silencing conditions. In contrast, CUL7 overexpression promoted tumour growth, invasion and migration. Gene set enrichment analysis (GSEA) and western blot analysis revealed that CUL7 was positively associated with the NF-κB pathway. Moreover, with coimmunoprecipitation assays, we discovered that CUL7 physically associated with MST1, which further led to ubiquitin-mediated MST1 protein degradation, which promoted activation of the NF-κB signalling pathway. Finally, CUL7 was found to be downregulated by miR-3940-5p, which suppressed the development of gliomas. CONCLUSIONS: Our findings indicate that CUL7 plays a significant role in promoting tumorigenesis via NF-κB activation and that it can be negatively regulated by miR-3940-5p in human gliomas. Furthermore, CUL7 might be a candidate molecular target for the treatment of glioma.

Methylation in the TP53 promoter is associated with ischemic stroke.

Cerebral ischemic stroke (IS) is a disease presenting high morbidity and mortality rates worldwide. Understanding of the pathogenesis underlying IS may facilitate the development of effective clinical therapeutic strategies and improve the prevention of this disease, decreasing its occurrence rate. Epigenetic alterations have recently attracted attention as possible mechanisms underlying IS. Additionally, tumor protein p53 (TP53) was identified to be involved in the pathophysiology of cerebral stroke. In the present study, the methylation status of the TP53 promoter was investigated in patients with IS and in age­matched healthy controls. The methylation status of the promoter of TP53 was significantly increased in patients with IS compared with healthy subjects. Additionally, the methylation level of the TP53 promoter was identified to be associated with carotid intima­media thickness, the degree of carotid atherosclerosis and the circulating levels of homocysteine in peripheral blood. The present findings may improve the understanding of the role of the epigenetic modifications of the TP53 promoter in IS pathogenesis.

Mucin O-glycosylating enzyme GALNT2 facilitates the malignant character of glioma by activating the EGFR/PI3K/Akt/mTOR axis.

N-Acetylgalactosaminyltransferase 2 (GALNT2), the enzyme that regulates the initial step of mucin O-glycosylation, has been reported to play a role in influencing the malignancy of various cancers. However, the mechanism through which it influences gliomas is still unknown. In the current study, the Cox proportional hazards model was used to select genes. Data obtained from The Cancer Genome Atlas (TCGA) database and immunohistochemistry (IHC) of clinical specimens showed that increased GALNT2 expression levels were associated with an unfavorable prognosis and a higher tumor grade in human gliomas. Then, GALNT2 knockdown and overexpression were performed in glioma cell lines and verified by quantitative real-time PCR (qRT-PCR) and Western blotting. Functional assays demonstrated that GALNT2 was closely related to glioma cell proliferation, cycle transition, migration and invasion. Western blot analysis and lectin pull-down assays indicated that GALNT2 knockdown decreased the level of phosphorylated epidermal growth factor receptor (EGFR) and the expression of the Tn antigen on EGFR and affected the expression levels of p21, cyclin-dependent kinase 4 (CDK4), cyclinD1, matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) through the EGFR/PI3K/Akt/mTOR pathway. GALNT2 overexpression had the opposite effects. In vivo, the growth of orthotopic glioma xenografts in nude mice was distinctly inhibited by the expression of GALNT2 shRNA, and the tumors with GALNT2 shRNA exhibited less aggressiveness and reduced expression of Ki67 and MMP2. Overall, GALNT2 facilitates the malignant characteristics of glioma by influencing the O-glycosylation and phosphorylation of EGFR and the subsequent downstream PI3K/Akt/mTOR axis. Therefore, GALNT2 may serve as a novel biomarker and a potential target for future therapy of glioma.