MiR-124 Reduced Neuroinflammation after Traumatic Brain Injury by Inhibiting TRAF6.

INTRODUCTION: Neuroinflammation contributes to secondary injury after traumatic brain injury (TBI), which has been mainly mediated by the microglia. MiR-124 was reported to play an important role in the polarization of microglia by targeting TLR4 signaling pathway. However, the role and mechanism of miR-124 in neuroinflammation mediated by microglia after TBI is unclear. To clarify this, we performed this research. METHODS: The expression of miR-124 was first measured by RT-PCR in the injured brain at 1/3/7 days post-TBI. Then, miR-124 mimics or inhibitors administration was used to interfere the expression of miR-124 at 24 h post-TBI. Subsequently, the microglia polarization markers were detected by RT-PCR, the expression of inflammatory cytokines was detected by ELISA, the expression of TLR4/MyD88/IRAK1/TRAF6/NF-κB was measured by WB, and the neurological deficit was evaluated by NSS and MWM test. At last, in vitro experiments were performed to explore the exact target molecule of miR-124 on TLR4 signaling pathway. RESULTS: Animal research indicated that the expression of miR-124 was downregulated after TBI. Upregulation of miR-124 promoted the M2 polarization of microglia and inhibited the activity of TLR4 pathway, as well as reduced neuroinflammation and neurological deficit after TBI. In vitro experiments indicated that miR-124 promoted the M2 polarization of microglia and reduced neuroinflammation by inhibiting TRAF6. CONCLUSION: This study demonstrated that upregulation of miR-124 promoted the M2 polarization of microglia and reduced neuroinflammation after TBI by inhibiting TRAF6.

Drp1-Mediated Mitochondrial Metabolic Dysfunction Inhibits the Tumor Growth of Pituitary Adenomas.

Metabolic changes have been suggested to be a hallmark of tumors and are closely associated with tumorigenesis. In a previous study, we demonstrated the role of lactate dehydrogenase in regulating abnormal glucose metabolism in pituitary adenomas (PA). As the key organelle of oxidative phosphorylation (OXPHOS), mitochondria play a vital role in the energy supply for tumor cells. However, few attempts have been made to elucidate mitochondrial metabolic homeostasis in PA. Dynamin-related protein 1 (Drp1) is a member of the dynamin superfamily of GTPases, which mediates mitochondrial fission. This study is aimed at investigating whether Drp1 affects the progression of PA through abnormal mitochondrial metabolism. We analyzed the expression of dynamin-related protein 1 (Drp1) in 20 surgical PA samples. The effects of Drp1 on PA growth were assessed in vitro and in xenograft models. We found an upregulation of Drp1 in PA samples with a low proliferation index. Knockdown or inhibition of Drp1 enhanced the proliferation of PA cell lines in vitro, while overexpression of Drp1 could reversed such effects. Mechanistically, overexpressed Drp1 damaged mitochondria by overproduction of reactive oxygen species (ROS), which induced mitochondrial OXPHOS inhibition and decline of ATP production. The energy deficiency inhibited proliferation of PA cells. In addition, overexpressed Drp1 promoted cytochrome c release from damaged mitochondria into the cytoplasm and then activated the downstream caspase apoptotic cascade reaction, which induced apoptosis of PA cells. Moreover, the decreased ATP production induced by Drp1 overexpressing activated the AMPK cellular energy stress sensor and enhanced autophagy through the AMPK-ULK1 pathway, which might play a protective role in PA growth. Furthermore, overexpression of Drp1 repressed PA growth in vivo. Our data indicates that Drp1-mediated mitochondrial metabolic dysfunction inhibits PA growth by affecting cell proliferation, apoptosis, and autophagy. Selectively targeting mitochondrial metabolic homeostasis stands out as a promising antineoplastic strategy for PA therapy.

Drp1 Regulated Mitochondrial Hypofission Promotes the Invasion and Proliferation of Growth Hormone-Secreting Pituitary Adenomas via Activating STAT3.

The invasiveness and high proliferation rate of growth hormone-secreting pituitary adenomas (GHPAs) are closely related to poor prognosis in patients. We previously reported that abnormal glycolysis participates in this process; however, the role of mitochondria in the invasion and proliferation of GHPAs remains unknown. In the current study, stereological methods were first used to quantitatively calculate the number and morphology of mitochondria. The results revealed that the numbers, volumes and membrane areas of mitochondria were decreased in invasive GHPAs (IGHPAs) samples compared to noninvasive GHPAs (NIGHPAs) samples. Furthermore, significantly downregulated mRNA and protein levels of dynamin-related protein 1 (Drp1) were detected in IGHPAs, but no notable changes in fusion related molecules (Mfn1, Mfn2 and OPA1) were detected, suggesting that the abnormal mitochondrial dynamics in IGHPAs are characterized by hypofission. Mitochondrial hypofission caused by Mdivi-1, a specific Drp1 inhibitor, enhanced the invasion and proliferation of GH3 cell lines and primary cells from patients with GHPAs in vitro and in vivo, while overexpression of Drp1 reversed these processes. Mechanistically, mitochondrial hypofission might activate signal transducer and activator of transcription 3 (STAT3). Specifically, elevated nuclear pSTAT3Y705 may promote GH3 cell invasion by upregulating the activity of matrix metalloproteinase 2/9, and elevated mitochondrial pSTAT3S727 may promote GH3 cell proliferation by inhibiting the mitochondria-dependent apoptotic pathway. Taken together, our findings suggest that mitochondrial hypofission induced by Drp1 might strengthen the invasion and proliferation of GHPA tumor cells by activating STAT3, providing us with a new perspective on how mitochondria regulate the development of IGHPAs.

SCP2-mediated cholesterol membrane trafficking promotes the growth of pituitary adenomas via Hedgehog signaling activation.

BACKGROUND: Metabolic reprogramming is an important characteristic of tumors. In the progression of pituitary adenomas (PA), abnormal glucose metabolism has been confirmed by us before. However, whether cholesterol metabolism is involved in the process of PA remains unclear. This study aimed to investigate whether abnormal cholesterol metabolism could affect the progression of PA. METHODS: We analyzed the expression of sterol carrier protein 2 (SCP2) in 40 surgical PA samples. In vitro experiments and xenograft models were used to assess the effects of SCP2 and cholesterol on proliferation of PA. The incidence of hypercholesterolemia between 140 PA patients and 100 heathy controls were compared. RESULTS: We found an upregulation of SCP2 in PA samples, especially in tumors with high proliferation index. Forced expression of SCP2 promoted PA cell lines proliferation in vitro. Furthermore, SCP2 regulated cholesterol trafficking from cytoplasm to membrane in GH3 cells, and extracellularly treating GH3 cells and primary PA cells with methyl-ß-cyclodextrin/cholesterol complex to mimic membrane cholesterol concentration enhanced cell proliferation, which suggested a proliferative effect of cholesterol. Mechanistically, cholesterol induced activation of PKA/SUFU/GLI1 signaling via smoothened receptor, which was well-known as Hedgehog signaling, resulting in inhibiting apoptosis and promoting cell cycle. Accordingly, activation of Hedgehog signaling was also confirmed in primary PA cells and surgical PA samples. In vivo, SCP2 overexpression and high cholesterol diet could promote tumor growth. Intriguingly, the incidence of hypercholesterolemia was significantly higher in PA patients than healthy controls. CONCLUSIONS: Our data indicated that dysregulated cholesterol metabolism could promote PA growth by activating Hedgehog signaling, supporting a potential tumorigenic role of cholesterol metabolism in PA progression.

Impact of interaction between the G870A and EFEMP1 gene polymorphism on glioma risk in Chinese Han population.

AIMS: To investigate the impact of CCND1 and EFEMP1 gene polymorphism, and additional their gene-gene interactions and haplotype within EFEMP1 gene on glioma risk based on Chinese population. METHODS: Logistic regression was performed to investigate association between single-nucleotide polymorphisms (SNP) and glioma risk and generalized multifactor dimensionality reduction (GMDR) was used to analyze the gene-gene interaction. RESULTS: Glioma risks were higher in carriers of homozygous mutant of rs603965 within CCND1 gene, rs1346787 and rs3791679 in EFEMP1 gene than those with wild-type homozygotes, OR (95%CI) were 1.67 (1.23-2.02), 1.59 (1.25-2.01) and 1.42 (1.15-1.82), respectively. GMDR analysis indicated a significant two-locus model (p=0.0010) involving rs603965 within CCND1 gene and rs1346787 within EFEMP1 gene. Overall, the cross-validation consistency of the two- locus models was 10\ 10, and the testing accuracy is 60.17%. Participants with rs603965 - GA or AA and rs1346787- AG or GG genotype have the highest glioma risk, compared to participants with rs603965 - GG and rs1346787- AA genotype, OR (95%CI) was 3.65 (1.81-5.22). We conducted haplotype analysis for rs1346787 and rs3791679, because D' value between rs1346787 and rs3791679 was more than 0.8. The most common haplotype was rs1346787 - A and rs3791679- G haplotype, the frequency of which was 0.4905 and 0.4428 in case and control group. CONCLUSIONS: Polymorphism in rs603965 within CCND1 gene and rs1346787 within EFEMP1 gene and its gene- gene interaction were associated with increased glioma risk.

NFAT5 protects astrocytes against oxygen-glucose-serum deprivation/restoration damage via the SIRT1/Nrf2 pathway.

Nuclear factor of activated T cells (NFAT) is a multifunctional cytokine family. NFAT5 was recently reported to be involved in many neuronal functions, but its specific function remains unclear. In this study, our aim is to investigate whether NFAT5 overexpression can protect astrocytes against oxygen-glucose-serum deprivation/restoration (OGSD/R) damage. In vivo, rats were subjected to ischemia-reperfusion injury, resulting in increased water content, infarct volume, and expression of NFAT5 protein in rat spinal cord. After primary culture for spinal cord astrocytes, the in vitro OGSD/R model was established. The results of the CCK8 assay and flow cytometry showed that, in the OGSD/R group, astrocyte cell viability was downregulated, but astrocyte apoptosis increased. Caspase 3 activity increased as well. Levels of NFAT5, as detected by real-time quantitative PCR and western blot, decreased under OGSD/R, as did SIRT1. Commercial kits for activity assays were used to show that OGSD/R inhibited SIRT1 activation but accelerated SOD activation after OGSD/R. Next, pcDNA-NFAT5 or NFAT5 siRNA was transfected into astrocytes. Overexpression of NFAT5 not only promoted the survival of the astrocytes and SIRT1 activation under OGSD/R but also inhibited cell apoptosis and SOD activation. Moreover, overexpression of NFAT5 apparently diminished histone acetylation and promoted the nuclear transport of Nrf2. Our results show that NFAT5 protects spinal astrocytes in a manner that depends on activation of the SIRT1/Nrf2 pathway. These findings present a novel potential molecular mechanism for NFAT5 therapy in the context of spinal cord injury.

Impact of Heat Shock Protein A 12B Overexpression on Spinal Astrocyte Survival Against Oxygen-Glucose-Serum Deprivation/Restoration in Primary Cultured Astrocytes.

Heat shock protein A 12B (HSPA12B) is a newly discovered member of the heat shock protein 70 family. Preclinical evidence indicates that HSPA12B helps protect the brain from ischemic injury, although its specific function remains unclear. The aim of this study is to investigate whether HSPA12B overexpression can protect astrocytes from oxygen-glucose-serum deprivation/restoration (OGD/R) injury. We analyzed the effects of HSPA12B overexpression on spinal cord ischemia-reperfusion injury and spinal astrocyte survival. After ischemia-reperfusion injury, we found that HSPA12B overexpression decreased spinal cord water content and infarct volume. MTT assay showed that HSPA12B overexpression increased astrocyte survival after OGD/R treatment. Flow cytometry results showed a marked inhibition of OGD/R-induced astrocyte apoptosis. Western blot assay showed that HSPA12B overexpression significantly increased regulatory protein B-cell lymphocyte 2 (Bcl-2) levels, whereas it decreased expression of the Bax protein, which forms a heterodimer with Bcl-2. Measurements of the level of activation of caspase-3 by Caspase-Glo®3/7 Assay kit showed that HSPA12B overexpression markedly inhibited caspase-3 activation. Notably, we demonstrated that the effects of HSPA12B on spinal astrocyte survival depended on activation of the PI3K/Akt signal pathway. These findings indicate that HSPA12B protects against spinal cord ischemia-reperfusion injury and may represent a potential treatment target.

Myocyte enhancer factor 2D promotes tumorigenicity in malignant glioma cells.

The prognosis of patients with malignant glioma is always quite poor, and this poor prognosis is probably due to our incomplete understanding of the molecular mechanisms underlying malignant glioma. It is known that myocyte enhancer factor-2D (MEF2D) plays an oncogenic role in hepatocellular carcinoma and promotes the survival of various types of cells. However, little is known about the expression profile and function of MEF2D in malignant glioma. In this study, we investigated the function and expression of MEF2D in malignant glioma. We found that in malignant glioma, there is an aberrantly high expression of MEF2D, which leads to poor prognosis of malignant glioma. The downregulation of MEF2D suppresses the proliferation of malignant glioma cell lines by inducing delay of S and G2/M phases of cell cycle and promoting apoptosis. Furthermore, the overexpression of MEF2D in astrocytes accelerates cell proliferation by regulating cell cycle progression. Furthermore, a mouse malignant glioma model demonstrated that MEF2D deficiency blocks malignant glioma formation in vivo. We conclude that MEF2D may act as a potential oncogene in malignant glioma and thus serve as a candidate target for malignant glioma therapy.

Glioma-derived thrombospondin-1 modulates cd14+ cell tolerogenic properties.

Thrombospondin-1 (TSP1) plays a role in the immune tolerance, and is involved in the pathogenesis of glioma. This study aims to investigate the role of the glioma-derived TSP1 in the induction of the tumor immune tolerance. The results showed that the primary human glioma cells expressed high levels of TSP1. Glioma cells enhanced the expression of transforming growth factor (TGF)-ß in CD4⁺ CD16⁻ naïve monocytes (Mos). The TGF-ß⁺ Mos showed inhibitory effect on CD8⁺ T cell proliferation. We conclude that glioma cell-derived TSP1 facilitates the induction of TGF-ß in Mos. The TSP1 may be a potential therapeutic target of glioma.

Analyzing time-series microarray data reveals key genes in spinal cord injury.

Although many scholars have utilized high-throughput microarrays to delineate gene expression patterns after spinal cord injury (SCI), no study has evaluated gene changes in raphe magnus (RM) and somatomotor cortex (SMTC), two areas in brain primarily affected by SCI. In present study, we aimed to analyze the differentially expressed genes (DEGs) of RM and SMTC between SCI model and sham injured control at 4, 24 h, 7, 14, 28 days, and 3 months using microarray dataset GSE2270 downloaded from gene expression omnibus and unpaired significance analysis of microarray method. Protein-protein interaction (PPI) network was constructed for DEGs at crucial time points and significant biological functions were enriched using DAVID. The results indicated that more DEGs were identified at 14 days in RM and at 4 h/3 months in SMTC after SCI. In the PPI network for DEGs at 14 days in RM, interleukin 6, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FBJ murine osteosarcoma viral oncogene homolog (FOS), tumor necrosis factor, and nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) were the top 5 hub genes; In the PPI network for DEGs at 3 months in SMTC, the top 5 hub genes were ubiquitin B, Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1), FOS, Janus kinase 2 and vascular endothelial growth factor A. Hedgehog and Wnt signaling pathways were the top 2 significant pathways in RM. These hub DEGs and pathways may be underlying therapeutic targets for SCI.

Emodin inhibits inducible nitric oxide synthase in a rat model of craniocerebral explosive injury.

To investigate the effects of emodin on blast-induced traumatic brain injury (bTBI) in a rat model. Eighty rats were randomly divided into 2 groups (the control group and the emodin-treated group; N = 40 per group) and were used to establish the model of blast-induced traumatic brain injury. Ten minutes after the explosion, an isotonic saline solution (10 mg/kg) or emodin (10 mg/kg) were administered via an intraperitoneal injection to the control group and the emodin-treated group, respectively. At each time point (pre-explosion, 2, 6, 12, 24 h after explosion), 2 rats were used for the pathological assessment and 6 rats were used for the biochemical assessment. The concentration of nitric oxide (NO) and the expression and activity of inducible nitric oxide synthase (iNOS) were measured at each time point by spectrophotometry and western blot analysis. Light and electron microscopy showed that the brain damage in the emodin-treated group was less serious than that observed in the control group. The concentration of NO in the emodin-treated group was lower compared with the control group (p < 0.05). Western blot analysis showed that protein expression in the emodin-treated group was lower than the control group (p < 0.05). Emodin can alleviate brain damage after bTBI by inhibiting iNOS. These findings suggest that emodin has a protective effect against bTBI. One possible mechanism may occur by inhibiting the expression and activity of iNOS and consequently decreasing the concentration of NO.

Prognostic value of coexistence of abnormal expression of micro-RNA-200b and cyclic adenosine monophosphate-responsive element-binding protein 1 in human astrocytoma.

Our aim was to investigate the expression of micro-RNA-200b (miR-200b) and cAMP-responsive element-binding protein 1 (CREB-1) in astrocytoma and its efficacy for predicting outcome. Both miR-200b and CREB-1 messenger RNA expression was measured in 122 astrocytomas and 30 nonneoplastic brain specimens by quantitative real-time polymerase chain reaction. Expression of miR-200b was significantly lower in astrocytoma than in nonneoplastic brain (P < .001), whereas CREB-1 messenger RNA expression was significantly elevated in the tumors (P < .001). Both miR-200b down-regulation and CREB-1 up-regulation were significantly associated with advanced pathologic grade (P = .002 and P = .006, respectively). Low miR-200b expression correlated negatively with Karnofsky performance score (P = .03), and high CREB-1 expression correlated positively with mean tumor diameter (P = .03). By Kaplan-Meier analysis, low miR-200b, high CREB-1, and coexistence of abnormal miR-200b and CREB-1 expression (low miR-200b/high CREB-1) were predictive of shorter progression-free survival and overall survival in both grade III and grade IV astrocytoma. By multivariate analysis, only low miR-200b/high CREB-1 expression was an independent prognostic factor for poor prognosis in astrocytoma of advanced grade. Both miR-200b and CREB-1 may play important cooperative roles in the progression of human astrocytoma. The efficacy of miR-200b and CREB-1 together as a predictor of prognosis in astrocytoma patients is shown for the first time.