Background: Subarachnoid hemorrhage (SAH) is a devastating disease which leads to high morbidity and mortality. Recent studies have indicated that, never in mitosis gene A-related expressed kinase 7 (NEK7), is involved in NLRP3 (NLR family, pyrin domain containing 3) associated inflammation, which may result in subsequent cellular and vascular damage. The aim of this study was to investigate whether NEK7 is involved in the pathophysiology of subarachnoid hemorrhage. Methods: 455 adult male C57B6J mice, weighing 22 to 30 g, were used to investigate the time course of NEK7 expression in the ipsilateral cortex after SAH, and to investigate the intrinsic function and mechanism of NEK7. A vascular puncture model was used to create the mouse SAH model, and intracerebroventricular injection was used to deliver NEK7 recombinant protein, NEK7 small interfering RNA, nigericin, and MCC950. Neurological score, brain water content, Evans blue extravasation, immunofluorescence, and western blot were evaluated for neurological outcome, neuronal apoptosis, blood-brain barrier damage, microglia accumulation, and the mechanism of NEK7 and NLRP3 activation. Results: Our results exhibited that intrinsic NEK7 was elevated after SAH in the cortex of the left/ipsilateral hemisphere and was colocalized with microglia, endothelial cells, neuron, astrocyte, and oligodendrocyte, and highly expressed in microglia and endothelial cells after SAH. NEK7 recombinant protein aggravated neurological deficits, brain edema, neuronal apoptosis, BBB permeability, microglial accumulation, and activated caspase-1 and IL-1β maturation, while NEK7 small interfering RNA injection reversed those effects. Nigericin administration enhanced ASC oligomerization, caspase-1 and IL-1β maturation without increasing the protein level of NLRP3, and ASC oligomerization and caspase-1 IL-1β maturation reduced when combined with NEK7 knockdown or MCC950 delivery. We found the level of NEK7 expression increased after SAH and could activate the downstream NLRP3 pathway to induce caspase-1, IL-1β expression and then increased the BBB opening, microglia accumulation and neuronal apoptosis after SAH. Conclusions: This study demonstrated for the first time that NEK7 mediated the harmful effects of neuronal apoptosis and BBB disruption after SAH, which may potentially be mediated by the NEK7/NLRP3 signal. NEK7 served as a co-component for NLRP3 inflammasome activation after SAH. NEK7 may be a promising target on the management of SAH patients.

BackgroundAurora kinase ensures accurate chromosome segregation during cell cycle, maintaining genetic integrity in cell division. VX-680, a small-molecule Aurora kinase inhibitor, interferes with mitotic entry and formation of bipolar spindles. Here, we evaluated VX-680 as a potential agent for treatment of all-trans retinoid acid (ATRA)-resistant acute promyelocytic leukemia (APL) in vitro.MethodsCD11b expression was utilized to assess cell differentiation by flow cytometry. Immunofluorescence staining was conducted to analyze formation of cell monopolar spindle. Cell proliferation was evaluated by MTT assay. Sub-G1 population and Annexin V/PI staining were used to measure cell apoptosis. Hoechst 33342 staining was applied for identifying morphological changes in nucleus of apoptotic cell. Aurora-A (Aur-A) activation and the signaling pathways involved in apoptosis were detected by Western blot. JC-1 probe was employed to measure mitochondrial depolarization.ResultsVX-680 inhibited Aur-A by reducing autophosphorylation at the activation site, Thr288, accompanied by producing monopolar mitotic spindles in APL cell line NB4-R2 that was resistant to ATRA. In addition, we found that VX-680 inhibited cell proliferation as assessed by MTT assay. Flow cytometry showed that VX-680 led to apoptotic cell death in both dose- and time-dependent manners by either Sub-G1 or Annexin V/PI analysis. Hoechst 33342 staining represented typical apoptotic cells with nuclear fragmentation in VX-680 treated cells. Importantly, VX-680 inhibition of Aurora kinase suppressed Akt-1 activation and induced mitochondrial depolarization, which eventually resulted in apoptosis by activation of caspase pathway, as indicated by increasing proteolytic cleavage of procaspase-3 and poly ADP ribose polymerase (PARP) in NB4-R2 cells.ConclusionsOur study suggested potential clinical use of mitotic Aurora kinase inhibitor in targeting ATRA-resistant leukemic cells.

Background: The mechanisms underlying the proliferation and apoptosis of glioma cells remain unelucidated. A recent study has revealed that microRNA-92b (miR-92b) inhibits apoptosis of glioma cells via downregulating DKK3. Notably, long noncoding RNA nuclear-enriched abundant transcript 1 (NEAT1) is predicted to have a possible interaction with miR-92b. Objective: This study aimed to identify whether NEAT1 affects glioma cell proliferation and apoptosis via regulating miR-92b. Methods: The expression of NEAT1 was compared between glioma tissues and adjacent tissues as well as between glioma cells and normal astrocytes using quantitative real-time polymerase chain reaction. Glioma cell proliferation was determined by using the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and glioma cell apoptosis was determined by using the flow cytometry. Results: The expression of NEAT1 was low in glioma tissues and cells compared to the normal ones. Overexpression of NEAT1 inhibited proliferation and promoted apoptosis of glioma cell lines (U-87 MG and U251). The interaction between NEAT1 and miR-92b was confirmed using RNA immunoprecipitation, RNA pull-down assay, and luciferase reporter assay. Importantly, the tumor suppressor function of overexpressing NEAT1 was achieved by downregulating miR-92b and subsequently upregulating DKK3. Conclusion: Our findings indicated that NEAT1 acts as a tumor suppressor in glioma cells, which provides a novel target in overcoming glioma growth.

No abstract

AIM: To determine whether melatonin (MLT) mitigates white matter (WM) injury by attenuating NOD-like receptor family pyrin domain-containing 3 (NLRP3)-associated oligodendrocyte apoptosis after subarachnoid hemorrhage (SAH). MATERIAL and METHODS: SAH model C57BL/6J mice were created using an endovascular perforation technique. The mice were injected intraperitoneally with MLT at doses of 50 mg/kg, 150 mg/kg and 300 mg/kg. The animals were evaluated for neurological outcomes according to neurological score, brain water content, myelin degradation, amyloid precursor protein (APP) accumulation, apoptosis, and levels of NLRP3, caspase-1, interleukin-1β, Bcl-2, and Bcl-2-interacting mediator of cell death (Bim) expression after SAH. RESULTS: MLT at a dose of 50 mg/kg improved neurological score, alleviated brain edema, and reduced WM injury. In addition, loss of myelin basic protein (MBP) and accumulation of APP, and expression in the ipsilateral/left hemisphere were found after SAH, and were reversed by MLT treatment. NLRP3 signal activation was found in microglia and apoptosis in oligodendrocytes were observed after SAH. MLT reduced oligodendrocyte apoptosis by regulating Bim and Bcl-2. CONCLUSION: Results of this study indicated that MLT exerts a WM-protective effect in SAH pathophysiology, possibly by attenuating apoptosis in oligodendrocytes.