LncRNA PVT1 promotes neuroinflammation after intracerebral hemorrhage by regulating the miR-128-3p/TXNIP axis.

OBJECTIVE: Intracerebral hemorrhage (ICH) has significant morbidity and mortality. TXNIP and the competing endogenous RNA (ceRNA) regulatory mechanism involved in long non-coding RNA (lncRNA) play roles in ICH. We probed the upstream microRNAs (miRNAs)/lncRNAs that regulated TXNIP expression in the ceRNA mechanism. METHODS: ICH mouse model was established, and ICH secondary injury was simulated in BV2 microglia by hemin treatment. TXNIP was silenced 48 h before ICH modeling. The ICH mouse brain water content (BWC) and brain lesion volume after ICH were recorded. Neuronal apoptosis and neurological deficits were evaluated by double staining of NeuN and TUNEL/modified Garcia/corner turn/forelimb placement tests. Iba1 + microglia number and tumor necrosis factor-α (TNF-α)/interleukin-1ß (IL-1ß)/IL-10/TXNIP/PVT1/miR-128-3p levels were assessed by immunohistochemistry, Western blot, ELISA, and RT-qPCR. Cell viability/death of BV2 cells conditioned medium-treated neuron HT22 cells were assessed by CCK-8/LDH assays. miRNA that had a targeted binding relationship with TXNIP was screened. The targeted bindings of miR-128-3p to TXNIP/PVT1 to miR-128-3p were verified by dual-luciferase reporter gene assay. RESULTS: TXNIP knockdown reduced post-ICH microglial activation/release of pro-inflammatory factors/brain edema/brain lesion volume/neurological deficits in mice and increased releases of anti-inflammatory factors. TXNIP/PVT1 knockdown inhibited hemin-induced inflammatory responses in BV2 cells and protected in vitro co-cultured HT22 cells. PVT1 was a sponge of miR-128-3p to repress TXNIP expression. miR-128-3p knockdown diminished PVT1 knockdown-inhibited hemin-induced BV2 cell inflammatory responses/neurotoxicity. CONCLUSIONS: PVT1 silencing reduced hemin-induced neuroinflammation and had a protective effect on neurons by increasing the targeted inhibition of TXNIP by miR-128-3p.

Nicotinic Acid-Mediated Modulation of Metastasis-Associated Protein 1 Methylation and Inflammation in Brain Arteriovenous Malformation.

We explored metastasis-associated protein 1 (MTA1) promoter methylation in the development of brain arteriovenous malformation (BAVM). The clinical data of 148 sex- and age-matched BAVMs and controls were collected, and the MTA1 DNA methylation in peripheral white blood cells (WBC) was assessed by bisulfite pyrosequencing. Among them, 18 pairs of case-control samples were used for WBC mRNA detection, 32 pairs were used for WBC MTA1 protein measurement, and 50 pairs were used for plasma inflammatory factor analysis. Lipopolysaccharide (LPS) treatment was used to induce an inflammatory injury cell model of human brain microvascular endothelial cells (BMECS). 5-Aza-2'-deoxycytidine (5-AZA), nicotinic acid (NA), and MTA1 siRNAs were used in functional experiments to examine BMECS behaviors. RT-qPCR, Western blot, and ELISA or cytometric bead arrays were used to measure the expression levels of MTA1, cytokines, and signaling pathway proteins in human blood or BMECS. The degree of MTA1 promoter methylation was reduced in BAVM compared with the control group and was inversely proportional to MTA1 expression. Plasma ApoA concentrations in BAVM patients were significantly lower than those in controls and correlated positively with MTA1 promoter methylation and negatively with MTA1 expression. The expression of cytokine was markedly higher in BAVM than in controls. Cell experiments showed that 5-AZA decreased the methylation level of MTA1 and increased the expression of MTA1 protein. LPS treatment significantly increased cytokine concentrations (p < 0.05). NA and MTA1 silencing could effectively reverse the LPS-mediated increase in IL-6 and TNF-α expression through the NF-κB pathway. Our study indicated that NA may regulate MTA1 expression by affecting promoter DNA methylation, improve vascular inflammation through the NF-κB pathway, and alleviate the pathological development of BAVM.

DNA Methylation of Patatin-Like Phospholipase Domain-Containing Protein 6 Gene Contributes to the Risk of Intracranial Aneurysm in Males.

Objective: This study is aimed to investigate the contribution of patatin-like phospholipase domain-containing protein 6 (PNPLA6) DNA methylation to the risk of intracranial aneurysm (IA) in the Han Chinese population. Methods: A total of 96 age- and sex-matched participants were recruited to evaluate PNPLA6 methylation via bisulfite pyrosequencing. The PNPLA6 mRNA expression in the plasma was determined using real-time quantitative reverse transcription-polymerase chain reaction. Human primary artery smooth muscle cells (HPCASMC) were used for the in vitro function study. Results: PNPLA6 methylation was significantly higher in patients with IA than in healthy controls (p < 0.01). Sex group analysis showed that this correlation appeared in the male group (p < 0.01) but not in the female group (p > 0.05). PNPLA6 methylation was significantly associated with age in all participants (r = 0.306, p = 0.003) and in the control group (r = 0.377, p = 0.008) but not in the IA group (r = 0.127, p = 0.402). Furthermore, the PNPLA6 mRNA expression significantly decreased in patients with IA than that in the controls (p = 0.016). PNPLA6 expression was significantly inversely correlated with elevated DNA methylation in participants (r = -0.825, p < 0.0001). In addition, PNPLA6 transcription was significantly enhanced following treatment with 5-aza-2'-deoxycytidine methylation inhibitor in HPCASMC.The receiver operating characteristic analyses of curves showed that the PNPLA6 mean methylation [area under the curve (AUC) = 0.74, p < 0.001] and mRNA expression (AUC = 0.86, p < 0.001) could have a diagnostic value for patients with IA. Conclusion: Although future functional experiments are required to test our hypothesis, our study demonstrated that PNPLA6 methylation and mRNA expression were significantly associated with the risk of IA; thus, they show potential for use in the early diagnosis of IA.

The RNA-binding protein fragile-X mental retardation autosomal 1 (FXR1) modulates glioma cells sensitivity to temozolomide by regulating ferroptosis.

Temozolomide (TMZ) is a first-line chemotherapeutic agent for the treatment of glioma. However, at least 50% of glioma patients do not respond to TMZ, and the exact mechanism leading to TMZ resistance is still unclear. In the present study, we investigated molecular mechanisms underlying the resistance to TMZ in glioma cells. Glioma cell lines A172 and U251 were maintained in medium with increasing doses of TMZ for 12 months to induce the TMZ-resistance. Cells were then transduced with different adenoviral vectors to overexpress or inhibit RNA-binding protein fragile-X mental retardation autosomal 1 (FXR1) and glutathione peroxidase 4 (GPX4), which has been associated with the ferroptosis mechanism. Cell viability and cell death were analysed using cell counting Kit-8 (CCK-8) and Annexin V-FITC staining, respectively. RT-PCR, RNA-seq analysis, and RNA immunoprecipitation were used to analyse RNA expression; Western blot was used for protein expression. We discovered that RNA-binding protein fragile-X mental retardation autosomal 1 (FXR1) was upregulated in TMZ-resistance glioma. Knockdown of FXR1 could overcome TMZ-resistance by promoting ferroptosis. Mechanically, FXR1 could bind with GPX4 mRNA and positively regulate the expression of GPX4. Inhibition of GPX4 further increased the sensitivity to TMZ in glioma cells with upregulated FXR1. Our data suggest that targeting FXR1-GPX4 might be a potential strategy to overcome chemoresistance to TMZ in glioma cells.

Study on the effect of shikonin on CD36 expression and phagocytic ability of microglia in the isolated cerebral haemorrhage model.

AIM OF THE STUDY: To investigate the effects of shikonin on CD36 expression and phagocytic ability of microglia, and its protective effect on neurons and the possible mechanism within. MATERIAL AND METHODS: The effects of shikonin on CD36 expression and phagocytic ability of microglia were detected by Western blot method, and cerebral haemorrhage was isolated by flow cytometry in the experiment. The protective effect of neurons was observed through neuron-microglia co-culture technique. Meanwhile, the effect of hydrogen peroxide on the expression of catalase was detected, and the concentration of hydrogen peroxide was measured in the isolated cerebral haemorrhage model. The t test was used to compare data between 2 groups, and one-way ANOVA was applied to multiple sets of data. RESULTS: Compared with the control group, the CD36 expression and phagocytic ability of microglia was increased by shikonin in the isolated cerebral haemorrhage model, while inflammatory factors such as tumour necrosis factor a (TNF-a) and interleukin 1b (IL-1b) attenuated the effects of the drug. The amount of neuron apoptosis/necrosis was significantly reduced by the drug, while the expression of catalase in microglia was increased, but the secretion of hydrogen peroxide was decreased in the neuron-microglia co-culture system. CONCLUSIONS: Shikonin can enhance the CD36 expression and the ability to phagocytose erythrocyte of microglia. Simultaneously, shikonin performs protective effects on neuronal cells and promotes the absorption of haematoma. Therefore, shikonin is probably an innovative medicine to treat cerebral haemorrhage.