Objective:To investigate the methylation status of O6-methylguanine-DNA methyltransferase (MGMT) in meningioma tissue and its effect on the growth and metastasis of human meningioma cell line IOMM-Lee cells.Methods:The specimens of 34 patients with meningioma were collected. Methylation-specific polymerase chain reaction (MSP) method was used to detect MGMT methylation in meningioma tissue and normal brain tissue; immunohistochemical staining was used to detect the expression of MGMT in meningioma tissue and normal brain tissue. The cultured human glioma cell line IOMM-Lee cells were divided into blank control group (normal cultured IOMM-Lee cells), negative control group (empty vector virus transfected IOMM-Lee cells) and RNAi lentivirus transfection group (transfected with RNAi lentivirus vector to down-regulate the expression of MGMT). The expression of MGMT in IOMM-Lee cells was silenced by RNAi technology. The expression levels of MGMT mRNA and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot. The proliferation activity of cells was detected by cell counting kit-8 (CCK-8) test, the colony forming ability was detected by cell clone formation test, and the invasion and migration ability of cells in each group were detected by Transwell test and cell scratch test.Results:The methylation of MGMT in meningioma tissue reached 88.23% (30/34). MGMT methylation was not detected in normal brain tissue; the staining intensity of MGMT in meningioma tissue was significantly higher than that in normal brain tissue. Compared with the blank control group and the negative control group, the relative expression of MGMT mRNA and protein in IOMM-Lee cells of the RNAi lentiviral transfection group were significantly decreased (all P<0.05). After 24, 48 and 72 hours of transfection, the proliferation activity of IOMM-Lee cells decreased significantly (all P<0.05), with reduced number of cell clone formation and cell invasion (all P<0.05). The rate of scar healing decreased significantly ( P<0.05). Conclusions:MGMT is mostly hypermethylated in human meningiomas. Silencing the expression of MGMT in meningiomas can inhibit the growth and metastasis of meningiomas.

Objective:To investigate the role of miR-145 in inflammatory response and immune regulation after traumatic brain injury(TBI).Methods:Male C57BL/6 mice were randomly divided into sham operation group(Sham),model group(TBI),TBI+NC agomir group and TBI+miR-145 agomir group.Modified Nerve Injury Severity Score(mNSS)was used to evaluate neurological function after trauma.MWM test was used toevaluates neurocognitive function of mice after TBI.Flow cytometry was used to detect the number of Tregs in tbrain tissue of each group of mice.ELISA was used to detect expressions of inflammatory cytokines in hippocampus of each group of mice.Immunohistochemistry was used to detect expression of activated microglia/macrophage Iba-1 in hippocampus of each group of mice.RT-qPCR was used to detect expressions of M1/M2 microglia/macrophage marker genes iNOS,CD11b,CD206 and Arg1.TUNEL staining and neuronal nuclear immunity double staining with fluorescent label(NeuN)were used to detect neuronal apoptosis.Results:Compared with Sham group,expression of miR-145 in hippocampus of mice in TBI group was significantly decreased,the neurological damage was increased,and percentage of Tregs in CD4+T cell population in brain tissue was decreased.Expression levels of IL-1β,IL-6,TNF-α,IL-4,IL-10 and TGF-β in hippocampus were significantly increased,the number of activated microglia/macrophage IBA-1 was increased,expression levels of iNOS CD11b,CD206 and Arg1 were significantly increased,and the neuronal apoptosis was increased.Notch1,p21 and Hes1 mRNA and protein levels were significantly increased(all P<0.05).Compared with TBI+NC agomir group,expression of miR-145 in hippocampus of mice in TBI+miR-145 agomir group was significantly increased,and neurological damage was reduced.Percentage of Tregs in CD4+T cell population in brain tissue was significantly increased,expressions of pro-inflammatory cytokines IL-1β,IL-6,and TNF-α were decreased,while anti-inflammatory cytokines IL-4,IL-10 and TGF-β were significantly increased in hippocampus.The number of activated microglia/macrophage IBA-1 was significantly decreased,expression levels of iNOS and CD11b were decreased,while expression levels of CD206 and Arg1 were significantly increased.mRNA and protein levels of Notch1,p21 and Hes1 were significantly reduced(all P<0.05).Conclusion:Overexpression of miR-145 promotes M2 polarization of microglia to regulate post-traumatic neuroinflammatory response and improve behavioral dysfunction by increasing Treg level,which may be mediated by Notch signaling pathway.

Objective : To investigate the effect and mechanism of exosome ( Exo) transported miR⁃223 on brain tissue injury and microglial activation in rats with traumatic brain injury ( TBI) . Methods : The miR⁃NC plasmid and miR⁃223 mimic plasmid were transfected into HEK293 cells by liposome method , and the expression level of miR⁃223 in the cells was determined by quantitative real⁃time PCR . Exo was extracted from transfected HEK293 cells and identified by transmission electron microscopy , nanoparticle tracking analysis and Western blot , the expression level of miR⁃223 in Exo was determined by quantitative real⁃time PCR . Forty SD rats were randomly divided into sham group , model group , NC⁃Exo group and miR⁃223 ⁃Exo group , with 10 rats in each group , TBI model was prepared by modified Feeney free fall method in all groups except sham group , rats in NC⁃Exo group and miR⁃223 ⁃Exo group were injected with cell⁃derived Exo transfected with miR⁃NC plasmid and cell⁃derived Exo transfected with miR⁃223 mimic plasmid via tail vein , respectively . Two weeks later , hematoxylin⁃eosin (HE) staining was used to observe the pathological changes of brain tissue in each group , Nissl staining was used to detect the changes and distribution of Nissl bodies in each group , enzyme⁃linked immunosorbent assay (ELISA) was used to measure the serum levels of tumor necrosis factor⁃α (TNF⁃α ) , interleukin⁃1β (IL⁃1β) and interleukin⁃6(IL⁃6) , immunofluorescence double staining was used to observe the expression of nod⁃like receptor family pyrin domain containing 3(NLRP3) and ionized calcium binding adaptor molecule 1 (Iba⁃1) , Western blot was used to detect the protein expression of NLRP3 , apoptosis⁃associated speck⁃like protein containing( ASC) and Caspase⁃1 . Results: After transfection , compared with control group and miR⁃NC group , the relative expression of miR⁃223 in miR⁃223 group significantly increased (P < 0. 05) . The isolated particles had typical Exo morphology , the peak particle size was about 120 nm , the Exo marker proteins CD9 , CD63 and CD81 were significantly overexpressed , and the relative expression of miR⁃223 significantly non of brain tissue in the miR⁃223 ⁃Exo group was improved , the morphology and number of Nissl bodies were re⁃increased (P < 0. 05) . Compared with the model group , the damage phenome stored , the levels of TNF⁃α , IL⁃1β and IL⁃6 in serum decreased ( P < 0. 05) , the intensity of NLRP3 and Iba⁃1 fluorescence staining in brain tissue decreased (P < 0. 05) , the relative protein expressions of NLRP3 , ASC and Caspase⁃1 in brain tissue were down⁃regulated (P < 0. 05) . Conclusion Exo operation of miR⁃223 can significant ly improve brain tissue injury and inhibit microglial activation in TBI rats , which may be related to the inhibition of NLRP3 .