The m6A methyltransferase METTL3 promotes LPS-induced microglia inflammation through TRAF6/NF-κB pathway.

OBJECTIVES: Microglia are the main effectors in the inflammatory process of the central nervous system. Once overactivated, microglia may release pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and IL-18, etc.) and accelerate neurodegeneration. Here, we aimed to explore the mechanism of how m6A methyltransferase METTL3 affects the inflammatory response of microglia, appropriately inhibiting the overactivation of microglia. MATERIALS AND METHODS: Lipopolysaccharide (LPS) was used to construct a cellular inflammation model in vitro. To evaluate the expression of METTL3 and inflammatory cytokines (IL-1ß, IL-6, TNF-α and IL-18) in cells, RT-PCR and ELISA were carried out. The related protein (TRAF6, NF-κB and I-κB) expression was examined adopting Western blot. Dot blot experiment was used to assess the effect of regulating METTL3 on the m6A level. Methylated RNA immunoprecipitation reaction was used to measure the effect of METTL3 on the m6A level of TRAF6 mRNA 3'-UTR. The co-immunoprecipitation experiment (IP) proved that METTL3 combines with TRAF6. RESULTS: In LPS-mediated microglial inflammation, METTL3 expression was increased, and the expression of inflammatory cytokines (IL-1ß, IL-6, TNF-α and IL-18) and inflammatory proteins (TRAF6 and NF-κB) were upregulated. METTL3 level was positively correlated with TRAF6, and the two proteins could bind to each other. Overexpression of METTL3 promoted the activation of the TRAF6-NF-κB pathway in an m6A-dependent manner, and inhibiting NF-κB attenuated METTL3-mediated microglial activation. CONCLUSION: METTL3 promotes LPS-induced microglial inflammation by activating the TRAF6-NF-κB pathway.

The Effect and Mechanism of lncRNA NR2F1-As1/miR-493-5p/MAP3K2 Axis in the Progression of Gastric Cancer.

BACKGROUND: LncRNA NR2F1-AS1 has been identified as an oncogene in some human tumors, such as breast cancer, nonsmall cell lung cancer, and esophageal squamous cell carcinoma. Nonetheless, whether NR2F1-AS1 is involved in the progression of gastric cancer (GC) remains unknown. METHODS: The expression patterns of NR2F1-AS1, MAP3K2, and miR-493-5p in GC tissues and cells were detected by RT-qPCR. The protein expression of MAP3K2 was assessed by the Western blotting assay. The MTT assay and flow cytometry were performed to measure cell proliferation and cell apoptosis in GC cells. The transwell assay was adopted to assess cell migration in GC cells. The relationship between NR2F1-AS1, MAP3K2, and miR-493-5p was verified by a dual-luciferase reporter assay. RESULTS: The increased NR2F1-AS1 and MAP3K2 expressions were discovered in GC tissues and cells compared with control groups. Knockdown of NR2F1-AS1 and MAP3K2 dramatically suppressed cell proliferation and migration, while it enhanced cell apoptosis in GC cells. In addition, NR2F1-AS1 was found to be a sponge of miR-493-5p, and MAP3K2 was a downstream gene of miR-493-5p. Moreover, the expression of MAP3K2 was notably reduced by miR-493-5p, and NR2F1-AS1 counteracted the inhibition of miR-493-5p. CONCLUSION: Thus, NR2F1-AS1 was verified to regulate GC cell progression by sponging miR-493-5p to upregulate MAP3K2 expression.

Monosialotetrahexosylganglioside sodium combined with hyperbaric oxygen on nervous system development and brain physiology in children with hypoxic ischemic encephalopathy.

To investigate the short-term and long-term clinical effects of monosialotetrahexosylganglioside sodium (GM1) combined with hyperbaric oxygen in neonatal hypoxic ischemic encephalopathy. A total of 80 children with hypoxic ischemic encephalopathy who were admitted to our hospital from January 2016 to March 2018 were selected and divided into the observation group and control group according to a random alphabet method, with 40 cases in each group. Neuron-specific enolase (NSE) and amplitude-integrated electroencephalogram (aEEG) were monitored after treatment in both groups, and the mental development index (MDI) and the psychomotor development index (PDI) of children were evaluated by the Bayley Scales of Infant Development (BSID) 12 months after discharge. The results showed that there were no significant differences in NSE levels and aEEG scores of children with mild severity between the two groups after treatment (P > 0.05). However, in both moderate and severe children, the NSE level and aEEG score in the observation group were significantly lower than that in the control group (P < 0.05). There were no statistically significant differences in MDI and PDI scores of children with mild severity between the two groups after 12 months of treatment (P > 0.05). However, in both moderate and severe children, the MDI and PDI scores in the observation group were significantly higher than that in the control group (P < 0.05). The incidence of adverse reactions in the observation group and the control group were 12.50% and 7.50%, respectively, (P = 0.640). The treatment of neonatal Neonatal hypoxic ischemic encephalopathy (HIE) with GM1 combined with hyperbaric oxygen can significantly improve the short-term and long-term nervous system development and brain physiology in children with moderate and severe HIE.

miR-135b-dependent downregulation of S100B promotes neural stem cell differentiation in a hypoxia/ischemia-induced cerebral palsy rat model.

Cerebral palsy (CP) is frequently caused by brain injury during pregnancy, delivery, or the immediate postnatal period. The differentiation potential of neural stem cell (NSC) makes them effective in restoring injured tissues and organs with minimal risks of side effects. In this study, we identified a novel microRNA-135b (miR-135b) in CP and investigated its functional role in mediating NSC differentiation. CP models were established in Wistar rats and validated with the Y-maze test. Gain- and loss-of-function experimentation was performed on CP rats. Then NSCs were isolated and the expression patterns of miR-135b and S100B were altered in NSCs. S100B exhibited high expression in the hippocampus tissues of CP models, which was targeted by miR-135b. miR-135b elevation or S100B silencing resulted in promoted NSC differentiation, alleviated brain injury, and inhibited NSC apoptosis in hippocampus tissues of CP rats. S100B downregulation targeted by miR-135b overexpression contributed to the inactivation of the signal transducer and activator of transcription-3 (STAT3) pathway, which promoted NSC differentiation and proliferation but inhibited NSC apoptosis. Our results highlight the suppressor role played by miR-135b in CP by inducing NSC differentiation via inactivation of S100B-dependent STAT3 pathway.

Long non-coding RNA and microRNA-675/let-7a mediates the protective effect of melatonin against early brain injury after subarachnoid hemorrhage via targeting TP53 and neural growth factor.

The objective of this study was to identify the protective effect of melatonin (MT) against early brain injury (EBI) following subarachnoid hemorrhage (SAH) and explore the underlying molecular mechanism. Real-time polymerase chain reaction (PCR) and luciferase assay were utilized to detect the effect of MT on H19 expression level, computation analysis and luciferase assay were conducted to the underlying mechanism of let-7a and miR-675. Real-time PCR, western blot analysis, immunohistochemistry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and flow cytometry analysis were performed to detect the effect of MT on H19, miR-675, let-7a, TP53, neural growth factor (NGF) levels, cell viability, and apoptosis status. Melatonin increased H19 expression level by enhancing H19 transcriptional efficiency in a concentration-dependent manner. MiR-675 and let-7a directly targeted P53 and NGF, respectively, and miR-675 reduced luciferase activity of wild-type but not mutant TP53 3'UTR. Meanwhile, let-7a suppressed luciferase activity of wild-type but not mutant NGF 3'UTR. H2O2 increased number of SA-b-gal, and while MT administration repressed the premature senescence. H2O2 obviously upregulated expressions of H19, miR-675, and NGF, and downregulated let-7a and TP53 levels; however, MT treatment reduced expressions of H19, miR-675, and NGF, and improved let-7a and TP53 levels. Treating with MT attenuated the neurological deficits and reduced the brain swelling. MT treatment repressed apoptosis of neurons caused by SAH. Levels of H19, miR-675, and NGF were much higher in the SAH + MT group, while there were even higher levels of H19, miR-675, and NGF in the SAH group than in the sham group; levels of let-7a and TP53 were much lower in the SAH + MT group, while they were even lower in the SAH group than in the sham group. Our study revealed that treatment with MT protected against EBI after SAH by modulating the signaling pathways of H19-miR-675-P53-apoptosis and H19-let-7a-NGF-apoptosis.

A rat pup model of cerebral palsy induced by prenatal inflammation and hypoxia.

Animal models of cerebral palsy established by simple infection or the hypoxia/ischemia method cannot effectively simulate the brain injury of a premature infant. Healthy 17-day-pregnant Wistar rats were intraperitoneally injected with lipopolysaccharide then subjected to hypoxia. The pups were used for this study at 4 weeks of age. Simultaneously, a hypoxia/ischemia group and a control group were used for comparison. The results of the footprint test, the balance beam test, the water maze test, neuroelectrophysiological examination and neuropathological examination demonstrated that, at 4 weeks after birth, footprint repeat space became larger between the forelimbs and hindlimbs of the rats, the latency period on the balance beam and in the Morris water maze was longer, place navigation and ability were poorer, and the stimulus intensity that induced the maximal wave amplitude of the compound muscle action potential was greater in the lipopolysaccharide/hypoxia and hypoxia/ischemia groups than in the control group. We observed irregular cells around the periventricular area, periventricular leukomalacia and breakage of the nuclear membrane in the lipopolysaccharide/hypoxia and hypoxia/ischemia groups. These results indicate that we successfully established a Wistar rat pup model of cerebral palsy by intraperitoneal injection of lipopolysaccharide and hypoxia.