RabGEF1 functions as an oncogene in U251 glioblastoma cells and is involved in regulating AKT and Erk pathways.

BACKGROUND: RabGEF1 is a guanine-nucleotide exchange factor for RAB-5, which plays an oncogenic role in certain human cancers. However, the function of RabGEF1 in glioma has not been studied. Here, we report that the down-regulation of RabGEF1 inhibits the proliferation and metastasis, and induces autophagy of U251 glioblastoma cells. METHODS: The expression of RabGEF1 in glioma and normal tissues were measured by immunohistochemistry. Four siRNAs targeting different sites of RabGEF1 were conducted and the interference efficiencies were verified by qRT-PCR assay. Western blot was used to detect the expression of interest proteins. Cell proliferation was detected using CCK-8 and clone formation assay. Cell migration and invasion were analyzed by scratch assay and transwell assay, respectively. Flow cytometry was used to detect cell cycle distribution and apoptosis. RESULTS: RabGEF1 was significantly up-regulated in human glioma tissues. RabGEF1 knockdown reduced cell viability, induced cell cycle arrest and apoptosis in U251 cells. Cell migration and invasion were also inhibited when RabGEF1 silencing. Mechanism studies showed that Cyclin D1 and CDK4/6 were significantly down-regulated when RabGEF1 silencing. p53 and caspase mediated apoptotic pathway was activated by down-regulation of RabGEF1. Moreover, RabGEF1 knockdown also induced autophagy in glioma cells. The investigation of AKT and Erk pathways suggested that phosphorylated AKT, p70S6K and phosphorylated Erk were all decreased when RabGEF1 silencing. CONCLUSION: In conclusion, our data suggest that RabGEF1 is up-regulated in human glioma and down-regulation of RabGEF1 inhibited cell proliferation and metastasis, and induced autophagy of U251 glioblastoma cells, which might be mediated by inactivation of AKT and Erk signaling pathways.

M2 bone marrow-derived macrophage-derived exosomes shuffle microRNA-21 to accelerate immune escape of glioma by modulating PEG3.

BACKGROUND: Growing studies have focused on the role of microRNA-21 (miR-21) in glioma, thus our objective was to discuss the effect of M2 bone marrow-derived macrophage (BMDM)-derived exosomes (BMDM-Exos) shuffle miR-21 on biological functions of glioma cells by regulating paternally expressed gene 3 (PEG3). METHODS: Seventy-one cases of human glioma tissues and 30 cases of non-tumor normal brain tissues were collected and stored in liquid nitrogen. PEG3 and miR-21 expression in glioma tissues was tested. The fasting venous blood of glioma patients and healthy control was collected and centrifuged, and then the supernatant was stored at - 80 °C refrigerator. The contents of interferon (IFN)-γ and transforming growth factor-ß1 (TGF-ß1) in serum were tested by ELISA. Glioma cells and normal glial cells were cultured to screen the target cells for further in vitro experiments. BMDM-Exos was obtained by ultra-high speed centrifugation and then was identified. BMDM-Exos was co-cultured with U87 cells to detect the biological functions. The fasting venous blood of glioma patients was extracted and treated with ethylene diamine tetraacetic acid-K2 anti-freezing, and then CD8+T cells were isolated. CD8+T cells were co-cultured with U87 cells to detect the CD8+T proliferation, cell cytotoxic activity, U87 cell activity, as well as IFN-γ and TGF-ß1 levels. Moreover, BALB/c-nu/nu mice was taken, and the human-nude mouse glioma orthotopic transplantation model was established with U87 cells, and then mice were grouped to test the trends in tumor growth. The brain of mice (fixed by 10% formaldehyde) was sliced to detect the expression of Ki67 and proliferating cell nuclear antigen (PCNA). The spleen of mice was taken to prepare single-cell suspension, and the percentage of T lymphocytes in spleen to CD8+T cells was detected. RESULTS: PEG3 expression was decreased and miR-21 expression was increased in glioma cells and tissues. Depleting miR-21 or restoring PEG3 suppressed growth, migration and invasion as well as accelerated apoptosis of glioma cells, also raised CD8+T proliferation, cell cytotoxic activity, and IFN-γ level as well as decreased U87 cell activity and TGF-ß1 level. BMDM-Exos shuttle miR-21 promoted migration, proliferation and invasion as well as suppressed apoptosis of glioma cells by reducing PEG3. Exosomes enhanced the volume of tumor, Ki67 and PCNA expression, reduced the percentage of CD8+T cells in glioma mice. CONCLUSION: BMDM-Exos shuffle miR-21 to facilitate invasion, proliferation and migration as well as inhibit apoptosis of glioma cells via inhibiting PEG3, furthermore, promoting immune escape of glioma cells.

Identification and validation of an autophagy-related signature for predicting survival in lower-grade glioma.

Abnormal levels of autophagy have been implicated in the pathogenesis of multiple diseases, including cancer. However, little is known about the role of autophagy-related genes (ARGs) in low-grade gliomas (LGG). Accordingly, the aims of this study were to assess the prognostic values of ARGs and to establish a genetic signature for LGG prognosis. Expression profile data from patients with and without primary LGG were obtained from The Cancer Genome Atlas (TCGA) and Genome Tissue Expression databases, respectively, and consensus clustering was used to identify clusters of patients with distinct prognoses. Nineteen differentially expressed ARGs were selected with threshold values of FDR < 0.05 and |log2 fold change (FC)| ≥ 2, and functional analysis revealed that these genes were associated with autophagy processes as expected. An autophagy-related signature was established using a Cox regression model of six ARGs that separated patients from TCGA training cohort into high- and low-risk groups. Univariate and multivariate Cox regression analysis indicated that the signature-based risk score was an independent prognostic factor. The signature was successfully validated using the TCGA testing, TCGA entire, and Chinese Glioma Genome Atlas cohorts. Stratified analyses demonstrated that the signature was associated with clinical features and prognosis, and gene set enrichment analysis revealed that autophagy- and cancer-related pathways were more enriched in high-risk patients than in low-risk patients. The prognostic value and expression of the six signature-related genes were also investigated. Thus, the present study constructed and validated an autophagy-related prognostic signature that could optimize individualized survival prediction in LGG patients.

Long non-coding RNA AC122108.1 promotes lung adenocarcinoma brain metastasis and progression through the Wnt/ß-catenin pathway by directly binding to aldolase A.

BACKGROUND: Brain metastasis (BM) is a major pathological subtype of lung adenocarcinoma (LAD), but the pathogenic mechanisms of BM remain unclear. The potential prognostic biomarkers and therapeutic targets for BM of LAD urgently need to be identified. AC122108.1 is a recently discovered new long non-coding ribonucleic acid (RNA). METHODS: AC122108 was found to be overexpressed in a LAD BM cell model, and upregulated in 64.52% of LAD BM tissues. AC122108 is an independent factor of BM during LAD development; however, the molecular mechanisms and clinical significance of AC122108.1 in LAD have not yet been established. Additionally, in vitro and in vivo experiments showed that the direct binding of AC122108.1 with aldolase A (ALDOA) enhanced the proliferation, apoptosis, invasiveness, migration, and metastasis of LAD cells. RESULTS: This RNA-protein complex decreased the stability of the ß-catenin destruction complex, leading to the accumulation of ß-catenin in the cytoplasm and ultimately its translocation into the nucleus to activate Wnt(wingless/integrated)/ß-catenin signaling. CONCLUSIONS: Overall, AC122108.1 promotes LAD BM and its progression through the Wnt/ß-catenin pathway by directly binding to ALDOA. This study provides insights into the regulatory mechanism of the LAD BM. AC122108.1 may serve as a potential therapeutic target and prognostic biomarker of LAD.

Gold-nanourchin seeded single-walled carbon nanotube on voltammetry sensor for diagnosing neurogenerative Parkinson's disease.

α-synuclein is a predominantly expressing neuronal protein for understanding the neurodegenerative disorders. A diagnosing system with aggregated α-synuclein encoded by SNCA gene is necessary to make the precautionary treatment against Parkinson's disease (PD). Herein, gold-nanourchin conjugated anti-α-synuclein antibody was desired as the probe and seeded on single-walled carbon nanotube (SWCN) integrated interdigitated electrode (IDE). The surface morphology of SWCN-modified IDE and gold urchin-antibody conjugates were observed under FESEM, FETEM and AFM, the existing elements were confirmed. Voltammetry analysis revealed that the limit of fibril-formed α-synuclein detection was improved by 1000 folds (1 fM) with gold-nanourchin-antibody modified surface, compared to the surface with only antibody (1 pM). Validating the interaction of α-synuclein by Enzyme-linked Immunosorbent Assay was displayed the detection limit as 10 pM. IDE has a good reproducibility and a higher selectivity on α-synuclein as evidenced by the interactive analysis with the control proteins, PARK1 and DJ-1.

Ursolic acid derivative induces apoptosis in glioma cells through down-regulation of cAMP.

The present study was designed to synthesize and evaluate ursolic acid hybrid compounds against glioma cells. Initial screening revealed that most of the synthesized compounds displayed better inhibitory effect on glioma cell proliferation compared to parent ursolic acid. The mechanism of inhibitory effect of the most potent compound 6d on glioma cells was investigated in detail. Treatment with compound 6d significantly (p < 0.001) reduced U251 and C6 cell proliferation at 48 h. The growth of U251 and C6 glioma cells was reduced to minimum level (17 and 21%) on treatment with 10 µM concentration of compound 6d. Treatment of the U251 cells with 10 µM concentration of compound 6d caused a significant (p < 0.05) inhibition of cAMP level. In U251 cell cultures treatment with compound 6d at 10 µM concentration enhanced proportion of apoptotic cells to 69.32% compared to 2.34% in the control cultures. The compound 6d treatment of U251 cells for 48 h caused arrest of cell cycle in the G0/G1 phase with consequent decrease of cell population in G2/M and S phases. The results from TEM showed that compound 6d treatment of U251 cells for 48 h caused blebbing of the cell membranes, chromatin condensation, appearance of foamy cytoplasmic material and autophagic vacuoles. The results from SEM revealed that compound 6d treatment of U251 cells caused a marked inhibition of microvilli and extensions on the cell surfaces. Thus present study demonstrates that compound 6d inhibits glioma cell growth, induces apoptosis and arrest cell cycle through metabolic pathway down-regulation. Therefore, compound 6d can be evaluated further for the treatment of glioma.