Enhancer-driven transcription of MCM8 by E2F4 promotes ATR pathway activation and glioma stem cell characteristics.

BACKGROUND: Glioma stem cells (GSCs) are responsible for glioma recurrence and drug resistance, yet the mechanisms underlying their maintenance remains unclear. This study aimed to identify enhancer-controlled genes involved in GSCs maintenance and elucidate the mechanisms underlying their regulation. METHODS: We analyzed RNA-seq data and H3K27ac ChIP-seq data from GSE119776 to identify differentially expressed genes and enhancers, respectively. Gene Ontology analysis was performed for functional enrichment. Transcription factors were predicted using the Toolkit for Cistrome Data Browser. Prognostic analysis and gene expression correlation was conducted using the Chinese Glioma Genome Atlas (CGGA) data. Two GSC cell lines, GSC-A172 and GSC-U138MG, were isolated from A172 and U138MG cell lines. qRT-PCR was used to detect gene transcription levels. ChIP-qPCR was used to detect H3K27ac of enhancers, and binding of E2F4 to target gene enhancers. Western blot was used to analyze protein levels of p-ATR and γH2AX. Sphere formation, limiting dilution and cell growth assays were used to analyze GSCs growth and self-renewal. RESULTS: We found that upregulated genes in GSCs were associated with ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) pathway activation, and that seven enhancer-controlled genes related to ATR pathway activation (LIN9, MCM8, CEP72, POLA1, DBF4, NDE1, and CDKN2C) were identified. Expression of these genes corresponded to poor prognosis in glioma patients. E2F4 was identified as a transcription factor that regulates enhancer-controlled genes related to the ATR pathway activation, with MCM8 having the highest hazard ratio among genes positively correlated with E2F4 expression. E2F4 bound to MCM8 enhancers to promote its transcription. Overexpression of MCM8 partially restored the inhibition of GSCs self-renewal, cell growth, and the ATR pathway activation caused by E2F4 knockdown. CONCLUSION: Our study demonstrated that E2F4-mediated enhancer activation of MCM8 promotes the ATR pathway activation and GSCs characteristics. These findings offer promising targets for the development of new therapies for gliomas.

Exosomal miR-214-5p Released from Glioblastoma Cells Modulates Inflammatory Response of Microglia after Lipopolysaccharide Stimulation through Targeting CXCR5.

BACKGROUND AND OBJECTIVE: Exosomes communicate inter-cellularly and miRNAs play critical roles in this scenario. MiR-214-5p was implicated in multiple tumors with diverse functions uncovered. However, whether miR-214-5p is mechanistically involved in glioblastoma, especially via exosomal pathway, is still elusive. Here we sought to comprehensively address the critical role of exosomal miR-214-5p in glioblastoma (GBM) microenvironment. METHODS: The relative expression of miR-214-5p was determined by real-time PCR. Cell viability and migration were measured by MTT and transwell chamber assays, respectively. The secretory cytokines were measured with ELISA kits. The regulatory effect of miR-214-5p on CXCR5 expression was interrogated by luciferase reporter assay. Protein level was analyzed by Western blot. RESULTS: We demonstrated that miR-214-5p was aberrantly overexpressed in GBM and associated with poorer clinical prognosis. High level of miR-214-5p significantly contributed to cell proliferation and migration. GBM-derived exosomal miR-214-5p promoted inflammatory response in primary microglia upon lipopolysaccharide challenge. We further identified CXCR5 as the direct target of miR-214- 5p in this setting. CONCLUSION: Overexpression of miR-214-5p in GBM modulated the inflammatory response in microglia via exosomal transfer.

Hippocampal synaptic and neural network deficits in young mice carrying the human APOE4 gene.

INTRODUCTION: Apolipoprotein E4 (APOE4) is a major genetic risk factor for late-onset sporadic Alzheimer disease. Emerging evidence demonstrates a hippocampus-associated learning and memory deficit in aged APOE4 human carriers and also in aged mice carrying human APOE4 gene. This suggests that either exogenous APOE4 or endogenous APOE4 alters the cognitive profile and hippocampal structure and function. However, little is known regarding how Apoe4 modulates hippocampal dendritic morphology, synaptic function, and neural network activity in young mice. AIM: In this study, we compared hippocampal dendritic and spine morphology and synaptic function of young (4 months) mice with transgenic expression of the human APOE4 and APOE3 genes. METHODS: Hippocampal dendritic and spine morphology and synaptic function were assessed by neuronal imaging and electrophysiological approaches. RESULTS: Morphology results showed that shortened dendritic length and reduced spine density occurred at hippocampal CA1 neurons in Apoe4 mice compared to Apoe3 mice. Electrophysiological results demonstrated that in the hippocampal CA3-CA1 synapses of young Apoe4 mice, basic synaptic transmission, and paired-pulse facilitation were enhanced but long-term potentiation and carbachol-induced hippocampal theta oscillations were impaired compared to young Apoe3 mice. However, both Apoe genotypes responded similarly to persistent stimulations (4, 10, and 40 Hz for 4 seconds). CONCLUSION: Our results suggest significant alterations in hippocampal dendritic structure and synaptic function in Apoe4 mice, even at an early age.

DNM3, p65 and p53 from exosomes represent potential clinical diagnosis markers for glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive and deadly primary brain cancer that arises from astrocytes and classified as grade IV. Recently, exosomes have been reported as an essential mediator in diverse cancer carcinogenesis and metastasis. However, their role in GBM is still unclear. In this study, we aimed to investigate whether blood exosomes can be potential clinical diagnostic markers for GBM. METHODS: We used a xenograft orthotopic mouse model to detect the differentially expressed genes in the brain and blood exosomes of original/recurrent GBM. RESULTS: We found that recurrent GBM had stronger growth capacity and lethality than original GBM in the mouse model. A gene microarray of original tumors and blood exosomes from GBM orthotopic xenografts results showed that DNM3, p65 and CD117 expressions increased, whereas PTEN and p53 expressions decreased in both original tumors and blood exosomes. In the recurrent GBM tumor model, DNM3 and p65 showed increased expressions, whereas ST14 and p53 showed decreased expressions in tumor and blood exosomes of the recurrent GBM mouse model. CONCLUSION: In summary, we found that DNM3, p65 and p53 had a similar trend in brain and blood exosomes both for original and recurrent GBM, and could serve as potential clinical diagnostic markers for GBM.

Exosomal miR-221 targets DNM3 to induce tumor progression and temozolomide resistance in glioma.

MicroRNA is an important regulator of glioblastoma. This study aims at validating microRNA-221 (miR-221) as a biomarker for glioblastoma, and understanding how miR-221 regulates glioblastoma progression. Using clinical samples, miR-221 expression was analyzed by quantitative reverse-transcriptase PCR (qPCR). SHG-44 cells were treated with anti-miR-221 or U87MG-derived exosomes followed by monitoring changes in cell viability, migration and temozolomide (TMZ) resistance. Bioinformatics approach was used to identify targets of miR-221. The interaction between miR-221 and its target, DNM3 gene, was studied with dual-luciferase reporter assay, Spearman's correlation analysis, and western blotting. To verify that RELA regulates miR-221 expression, RELA-expressing vector or shRNA was introduced into SHG-44 cells and its effect on miR-221 expression was monitored. Both tissue-level and exosomal miR-221 expression increased with glioma grades. In SHG-44 cells, downregulating miR-221 expression inhibited cell proliferation, migration, and TMZ resistance, whereas incubation with U87MG-derived exosomes exerted tumor-promoting effects. DNM3 gene is a target of miR-221. RELA induced miR-221 expression. In glioma, elevated miR-221 expression is a biomarker for glioma. DNM3 is a target of miR-221 and RELA regulates miR-221 expression. The RELA/miR-221 axis is a target for glioma diagnosis and therapy.

Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injury.

Neuronal apoptosis is mediated by intrinsic and extrinsic signaling pathways such as the membrane-mediated, mitochondrial, and endoplasmic reticulum stress pathways. Few studies have examined the endoplasmic reticulum-mediated apoptosis pathway in the penumbra after traumatic brain injury, and it remains unclear whether endoplasmic reticulum stress can activate the caspase-12-dependent apoptotic pathway in the traumatic penumbra. Here, we established rat models of fluid percussion-induced traumatic brain injury and found that protein expression of caspase-12, caspase-3 and the endoplasmic reticulum stress marker 78 kDa glucose-regulated protein increased in the traumatic penumbra 6 hours after injury and peaked at 24 hours. Furthermore, numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells in the traumatic penumbra also reached peak levels 24 hours after injury. These findings suggest that caspase-12-mediated endoplasmic reticulum-related apoptosis is activated in the traumatic penumbra, and may play an important role in the pathophysiology of secondary brain injury.

[Changes of protein kinase C activity in experimental presyrinx state in rabbits].

OBJECTIVE: To investigate the changes of protein kinase C (PKC) activity and its role in the development of presyrinx state in rabbits. METHODS: Presyrinx state was established in 56 rabbits by intra-cisternal injection of kaolin. At 1, 3, 7, 14, and 21 days after the injection, the water content in the upper cervical spinal cord was measured, its pathological changes observed microscopically and the PKC activity determined with substrate phosphorolysis kinase assay. RESULTS: Spinal cord edema occurred in rabbits one day after kaolin injection, with water content of (68.35-/+0.70)%, which increased to (72.70-/+0.88)% on day 3, reaching the peak level of (72.92-/+0.86)% on day 7, followed by gradual decline after 3 weeks [(70.03-/+0.77)%]. The membrane PKC activity increased from 5.67-/+0.26 pmol.mg(-1).min(-1) on day 1 after the injection to reach the peak level on day 7 (13.27-/+3.15 pmol.mg(-1).min(-1)), which was maintained till day 14 with subsequent decrease to 8.85-/+1.56 pmol.mg(-1).min(-1) on day 21. The cytoplasmic PKC activity showed changes of a reverse pattern. CONCLUSION: In rabbits with experimental presyrinx state, PKC translocation and activation is involved in ischemic spinal edema.

[Expression of aquaporin 4 during development of experimential presyrinx state in rabbits].

OBJECTIVE: To investigate the expression of AQP4 during the development of presyrinx state of experimental syringomyelia in rabbits. METHODS: The experimental syringomyelia models of rabbits were established by intra-cisternal injection of Kaolin. The expression of AQP4 AQP4mRNA and the water content of upper cervical spinal cord were measured with immunohistochemistry Western blot RT-PCR and dry-wet measurement on days 1,3,7,14, and 21 after operation, respectively. RESULTS: Compared with animals of control group, the water content increased in those of Kaolin group from the 1st day (68.35%+/-0.70%), reached its peak on the 7th day (72.92%+/-0.86%), lasted to the 14th day (72.58%+/-0.55%), and then began to drop on the 21st day (70.03%+/-0.77%), while AQP-4 immunoreactive expression decreased on the 3rd day [integral optical density(IOD) 320.5+/-44.2], reached its minimum on the 7th day (IOD 258.7+/-26.5), lasted to the 14th day, and recovered partially on the 21st day approximately (IOD 321.5+/-46.1). RT-PCR found the decreasing of AQP4 mRNA coincided well with that of AQP4 immunoreactive expression in presyrinx state. The linear regression analysis indicated that expression of AQP4 and its mRNA in cervical cord had a negative correlation with the change of spinal water content (r=-0.769, P<0.01; r=-0.955, P<0.01). CONCLUSION: Downregulation of AQP4 and its mRNA expression may involve in edema formation in the presyrinx state of rabbits.