Circular RNA circFAT1(e2) Facilitates Cell Progression through the miR-30e-5P/MYBL2 Pathway in Glioma.

Objective: To explore the mechanism of glioma from MYB family genes from the perspective of the circRNA-miRNA-mRNA regulatory network. Methods: First, the MYB family genes were analyzed by multiple bioinformatics analyses to identify one gene most associated with glioma. Then, the prognostic value and clinical characteristics of the gene were evaluated by bioinformatics analysis and experiments in glioma cells. Next, the target miRNA and circRNA were predicted and verified by dual-luciferase reporter assays. Besides, the functions of target circRNA in glioma were investigated by CCK-8 and Transwell assays. At last, the relation between the screened MYB gene, miRNA, and circRNA in glioma was identified by rescue experiments. Results: After expression and Cox and survival analysis of six MYB family genes, MYBL2 was identified as the gene most associated with glioma. Then, we found that MYBL2 expression in primary gliomas was higher than those in other histologies, and it had variable expression according to clinical features. Furthermore, MYBL2 knockdown in glioma cells impairs cell growth, invasion, and migration in functional studies. Then, miR-30e-5p and circFAT1(e2) were identified as targets of MYBL2 by bioinformatics prediction and experimental verification. Finally, the relationship between circFAT1(e2), MYBL2, and miR-30e-5p was elucidated by rescue experiments. Conclusion: circFAT1(e2) could promote glioma development by regulating MYBL2 and miR-30e-5p, and MYBL2 has diagnostic and prognostic values in glioma.

ZNF655 Promotes the Progression of Glioma Through Transcriptional Regulation of AURKA.

Objectives: Glioma has a high degree of malignancy, strong invasiveness, and poor prognosis, which is always a serious threat to human health. Previous studies have reported that C2H2 zinc finger (ZNF) protein is involved in the progression of various cancers. In this study, the clinical significance, biological behavior, and molecule mechanism of ZNF655 in glioma were explored. Methods: The expression of ZNF655 in glioma and its correlation with prognosis were analyzed through public datasets and immunohistochemical (IHC) staining. The shRNA-mediated ZNF655 knockdown was used to explore the effects of ZNF655 alteration on the phenotypes and tumorigenesis of human glioma cell lines. Chromatin immunoprecipitation (ChIP)-qPCR and luciferase reporter assays were performed to determine the potential mechanism of ZNF655 regulating Aurora kinase A (AURKA). Results: ZNF655 was abundantly expressed in glioma tissue and cell lines SHG-44 and U251. Knockdown of suppressed the progression of glioma cells, which was characterized by reduced proliferation, enhanced apoptosis, cycle repression in G2, inhibition of migration, and weakened tumorigenesis. Mechanistically, transcription factor ZNF655 activated the expression of AURKA by directly binding to the promoter of AURKA. In addition, downregulation of AURKA partially reversed the promoting effects of overexpression of ZNF655 on glioma cells. Conclusions: ZNF655 promoted the progression of glioma by binding to the promoter of AURKA, which may be a promising target for molecular therapy.

LncRNA MEG3 regulates microglial polarization through KLF4 to affect cerebral ischemia-reperfusion injury.

This study aimed to explore whether long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) affects the polarization of microglia in cerebral ischemia-reperfusion (I/R) injury through regulating Krüppel-like factor 4 (KLF4). A middle cerebral artery occlusion/reperfusion-induced (MCAO/R-induced) mouse model was established as an in vivo model. Oxygen and glucose confinement/reoxygenation-induced (OGD/R-induced) microglia (BV2 cells) were used as an in vitro model. RNA pull-down and RNA immunoprecipitation were used to detect the binding between MEG3 and KLF4. The MEG3 expression was signally elevated in the MCAO/R-induced mice or OGD/R-induced BV2 cells. The inhibition of MEG3 reversed the effects of OGD/R injury on the polarization and inflammation of BV2 cells. Moreover, MEG3 bound to KLF4 and inhibited its protein expression. Furthermore, the overexpression of MEG3 promoted M1 polarization and inflammation but inhibited M2 polarization by inhibiting KLF4 in BV2 cells. The transfection of small interfering RNAs against MEG3 inhibited M1 polarization and inflammation and promoted M2 polarization in vitro and in vivo. Inhibition of MEG3 can alleviate cerebral I/R injury via regulating the polarization of microglia through KLF4.NEW & NOTEWORTHY To study the role of long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) in cerebral ischemia-reperfusion (I/R) injury, we clarified the mechanism by which lncRNA MEG3 regulates the secretion of inflammatory cytokines in microglia through in vitro and in vivo experiments. We discovered that inhibition of MEG3 could alleviate cerebral I/R injury via inhibiting M1 polarization and promoting M2 polarization through Krüppel-like factor 4 (KLF4), indicating an effective theoretical basis for potential therapeutic targets of cerebral I/R injury.

MiR-367 alleviates inflammatory injury of microglia by promoting M2 polarization via targeting CEBPA.

MiR-367 was reported to regulate inflammatory response of microglia. CCAAT/enhancer-binding protein α (C/EBPA) could mediate microglia polarization. In this study, we explored the possible roles of miR-367 and CEBPA in intracerebral hemorrhage (ICH). ICH and normal specimens were obtained from the tissue adjacent to and distant from hematoma of ICH patients, respectively. Microglia were isolated and identified by immunofluorescence. The isolated microglia were treated with erythrocyte lysate and randomly divided into 8 groups using different transfection reagents. The transfection efficiency of miR-367 was determined by qRT-PCR. The expressions of M1 and M2 microglia markers were detected by Western blotting. The relationship between CEBPA and miR-367 was confirmed by dual luciferase reporter system. Flow cytometry was performed to determine the level of apoptosis in the cells transfected with miR-367 and CEBPA in erythrocyte lysate-treated microglia. We found that miR-367 expression level was downregulated in ICH specimens. Erythrocyte lysate-treated microglia was successfully established using erythrocyte lysate, as decreased miR-367 expression was observed. Overexpression of miR-367 could significantly decrease the expressions of MHC-ІІ, IL-1ß, and Bax, reduced apoptosis rate, and increased the expressions of CD206, Bal-2, and Arg-1 in erythrocyte lysate-treated microglia. CEBPA was proved to be a direct target for miR-367, which could inhibit microglia M2 polarization and increase apoptosis rate. However, in the presence of both CEBPA and miR-367 mimic, the protein and mRNA expressions of CEBPA were decreased, leading to promoted microglia M2 polarization and a decreased apoptosis rate. MiR-367 regulates microglia polarization by targeting CEBPA and is expected to alleviate ICH-induced inflammatory injury.

Circular RNA CircHIPK3 Elevates CCND2 Expression and Promotes Cell Proliferation and Invasion Through miR-124 in Glioma.

As a malignant tumor of the central nervous system, glioma exhibits high incidence and poor prognosis. Circular RNA HIPK3 (circHIPK3) is a circular RNA (circRNA) related to cancer progression. However, the role of circHIPK3 in gliomas remains unclear. The purpose of this study was to investigate the role of circHIPK3 in gliomas and its mechanism. The qRT-PCR method was used to determine the expression pattern of circHIPK3 in glioma cell lines. CCK-8 assay was used to detect cell proliferation. Cell migration and invasion were evaluated using the Transwell assay. Our results showed that circHIPK3 expression was significantly up-regulated in glioma tissues and cell lines. In vitro, the down-regulation of circHIPK3 significantly inhibited the proliferation, migration and invasion of glioma cells. Besides, we demonstrated that circHIPK3 acted as a sponge to absorb miR-124 and promoted CCND2 expression. In summary, our results indicated that circHIPK3 had carcinogenic effects by regulating the expression of CCND2 in glioma by sponging miR-124. These findings provided favorable evidence to reveal the role of circHIPK3 in the development of gliomas.

Long Noncoding RNA PCED1B-AS1 Promotes the Warburg Effect and Tumorigenesis by Upregulating HIF-1α in Glioblastoma.

Accumulating evidence suggests that long noncoding RNA (lncRNA) functions as a critical regulator in cancer biology. Here, we characterized the role of lncRNA PCED1B antisense RNA 1 (PCED1B-AS1) in glioblastoma (GBM). PCED1B-AS1 was notably upregulated in GBM tissues and cell lines and closely associated with larger tumor size and higher grade. Patients with high PCED1B-AS1 had shorter survival time than those with low PCED1B-AS1. Functional experiments showed that depletion of PCED1B-AS1 significantly inhibited, while overexpression of PCED1B-AS1 promoted cell proliferation, glucose uptake, and lactate release. Mechanistically, PCED1B-AS1 was able to directly bind to the 5'-UTR of HIF-1α mRNA and potentiate HIF-1α translation, leading to increased HIF-1α protein level, thereby promoting the Warburg effect and tumorigenesis. Importantly, PCED1B-AS1 lost the carcinogenic properties in the absence of HIF-1α. In addition, we also confirmed the existence of the PCED1B-AS1/HIF-1α regulatory axis in vivo. Taken together, our findings demonstrate that PCED1B-AS1 is a novel oncogenic lncRNA in GBM and functions in a HIF-1α-dependent manner, which provides a promising prognostic biomarker and druggable target for GBM.

Cinnamic acid rescues behavioral deficits in a mouse model of traumatic brain injury by targeting miR-455-3p/HDAC2.

AIMS: Traumatic brain injury (TBI) not only induces physiological disabilities but also leads to cognitive impairment. However, no effective therapeutic approach for TBI-related memory decline exists. In this study, we treated TBI mice with cinnamic acid (CNA) to detect whether CNA is able to rescue the memory deficits induced by TBI and to explore the potential mechanisms. MAIN METHODS: Mice were divided into the following groups: the sham group, the TBI group, the TBI + CNA group and the CNA group. Basic physiological parameters, neurological severity score and brain water content were analyzed. The Morris water maze and inhibitory avoidance step-down task were used to determine learning and memory. Golgi staining was used to measure alterations in dendritic spines. Western blot analysis and a commercial kit were used to detect the content and activity of HDAC2. qPCR was used to detect the relative level of miR-455. KEY FINDINGS: CNA did not affect physiological function but effectively restored neurological function and brain edema. CNA alleviated the memory impairments induced by TBI in both the Morris water maze and step-down task. CNA also recovered abnormalities in the synapses of TBI mice by suppressing the activity of HDAC2. Furthermore, CNA did not alter HDAC mRNA because it promoted the expression of miR-455-3p, a miRNA that regulates HDAC2 at the posttranscriptional level. SIGNIFICANCE: The application of CNA effectively treats TBI-induced memory deficits by increasing miR-455-3p and by inhibiting HDAC2.

Knockdown of NEAT1 repressed the malignant progression of glioma through sponging miR-107 and inhibiting CDK14.

Aberrant expressions of long noncoding RNAs (lncRNAs) contribute to carcinogenesis via regulating tumor suppressors or oncogenes. LncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been recognized as an oncogene to promote tumor progression of many cancers. However, the function of NEAT1 in glioma remains poorly discovered. Currently, we focused on the role of NEAT1 in glioma. Here, we found that NEAT1 was greatly upregulated in glioma cells compared with normal human astrocytes (NHAs). Meanwhile, miR-107 was significantly downregulated in glioma cell lines. Then, we observed that knockdown of NEAT1 suppressed the growth and invasion of glioma cells including U251 and SW1783 cells. Reversely, overexpression of NEAT1 dramatically induced glioma cell survival, increased cell colony formation, and promoted cell invasion ability. Subsequently, bioinformatics analysis was performed to predict the correlation between NEAT1 and miR-107. Moreover, it was revealed that NEAT1 could modulate miR-107 via serving as an endogenous sponge of miR-107. The direct binding correlation between NEAT1 and miR-107 was validated in our study. In addition, cyclin dependent kinase 14 (CDK14) was predicted as an messenger RNA target of miR-107 and the association between them was confirmed in our research. Moreover, we implied that NEAT1 demonstrated its biological functions via regulating miR-107 and CDK14 in vivo. In summary, our findings indicated that NEAT1/miR-107/CDK14 axis participated in glioma development. NEAT1 could act as a significant prognostic biomarker in glioma progression.

Design, synthesis and antiproliferative evaluation of novel sulfanilamide-1,2,3-triazole derivatives as tubulin polymerization inhibitors.

Microtubule as an important target in the cancer therapy was used to design novel tubulin polymerization inhibitors. Sulfanilamide-1,2,3-triazole hybrids were designed by a molecular hybridization strategy and their antiproliferative activity against three selected cancer cell lines (BGC-823, MGC-803 and SGC-7901) were evaluated. All sulfanilamide-1,2,3-triazole hybrids displayed potent inhibitory activity against all cell lines. In particular, compound 10b showed the most excellent inhibitory effect against MGC-803 cells, with an IC50 value of 0.4 µM. Cellular mechanism studies elucidated that 10b induced apoptosis by decreasing the expression level of Bcl-2 and Parp and increasing the expression level of BAX. 10b inhibited the epithelial-mesenchymal transition process by up-regulating E-cadherin and down-regulating N-cadherin. Furthermore, the tubulin polymerization inhibitory activity in vitro of 10b was 2.4 µM. In vivo anticancer assay, 10b effectively inhibited MGC-803 xenograft tumor growth without causing significant loss of body weight. These sulfanilamide-1,2,3-triazole hybrids as potent tubulin polymerization inhibitors might be used as promising candidates for cancer therapy.

A comparative CFD analysis of common carotid fusiform aneurysm in canine models and vertebrobasilar fusiform aneurysm in human patients.

BACKGROUND: Intracranial fusiform aneurysm (IFA) is a relatively uncommon subgroup of aneurysms. There are few reports that focus on the hemodynamics of IFA. In this study, we compared the hemodynamics of the canine model of common carotid fusiform aneurysm and vertebrobasilar fusiform aneurysms in human patients. METHODS: Five male mongrel dogs were randomly chosen, and their unilateral common carotid artery (CCA) and external jugular vein were surgically exposed individually. The CCA was transected and interposed by the free segment of the external jugular vein through end-to-end anastomosis to form a fusiform aneurysm. 3D digital subtraction angiography data of each dog's and five randomly chosen male patients' vertebrobasilar fusiform aneurysms were obtained and then analyzed by computational fluid dynamics software. The morphological and hemodynamic parameters were compared between the dogs and the patients. RESULTS: The morphological and hemodynamic parameters of the fusiform aneurysms were similar between the dogs and the patients. However, the hemodynamics was more complex in the patients. CONCLUSIONS: The canine fusiform aneurysm model exhibits high similarity in morphology and hemodynamics with human patients'. Therefore, this model can be used to study the fluid-solid interaction in the aneurysm and to explore the underlying mechanisms of the development, rupture and occurrence of IFAs, which offers a pathophysiological tool to seek better treatments of IFAs.

Geranylgeranylacetone exerts neuroprotective roles through medicating the phosphatidylinositol-3 kinase/Akt signaling pathway in an intracerebral hemorrhage rat model.

AIM: Previous studies have demonstrated that geranylgeranylacetone exerts neuroprotective effects in experimental intracerebral hemorrhage. This study is designed to explore the underlying mechanism. MATERIALS AND METHODS: One hundred and eighty male Sprague-Dawley rats were subjected to intracerebral hemorrhage by stereotactic injection of collagenase and were pretreated without or with different doses of geranylgeranylacetone. At 6 h, 24 h, 48 h, 72 h and 7 days after the operation, the neurological deficits were examined with the scoring scale method. To explore the underlying mechanism, wortmannin (Wort), a specific phosphatidylinositol-3 kinase (PI3K) inhibitor, was used. The protein expression of Akt was determined by Western blotting. The brain water content and the hematoma volume assessment were measured and compared among the different groups. RESULTS: We first found that geranylgeranylacetone pretreatment significantly reduced neurological deficit in intracerebral hemorrhage rats, indicating its neuroprotective role. Then, we found wort treatment significantly decreased the geranylgeranylacetone-induced Akt expression level in intracerebral hemorrhage rats. Besides, wort not only reversed the effects of geranylgeranylacetone on neurological function, but also reversed the effects of geranylgeranylacetone on reducing brain edema and decreasing hematoma volume in intracerebral hemorrhage rats. CONCLUSION: Geranylgeranylacetone exerts neuroprotective roles, at least partially, through medicating the PI3K/Akt signaling pathway in an experimental intracerebral hemorrhage rat model.

Transplantation of bone mesenchymal stem cells promotes angiogenesis and improves neurological function after traumatic brain injury in mouse.

Traumatic brain injury (TBI) has emerged as a leading cause of mortality and morbidity worldwide. Transplantation of bone mesenchymal stem cells (BMSCs) has emerged as a promising treatment for various central nervous system diseases. This study aims to evaluate the effect of BMSCs transplantation by intravenous injection on neurological function and angiogenesis of the TBI mice. C57BL/6 male mice were randomly divided into four groups: control, sham, TBI, and BMSC. Functional neurological evaluation was performed 1, 4, 7, 14, and 21 days after TBI using neurological severity scores. The impairment of learning and memory in mice was evaluated 14 days after TBI by Morris water maze experiment. Mice were sacrificed 14 days after TBI, and then brain sections were stained by terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling staining to assess brain neuronal apoptosis. Immunohistochemistry was conducted to evaluate caspase-3 activity and identify vascular distribution and microvessel density. Protein and mRNA levels of vascular endothelial growth factor (VEGF) and angiogenin-1 (Ang-1) in brain tissues were analyzed by Western blot and quantitative real-time polymerase chain reaction, respectively. BMSCs transplantation promoted recovery of neurological function, ameliorated impairment of learning and memory, attenuated neuronal apoptosis, and diminished caspase-3 activation in mice after TBI. Also, BMSCs transplantation upregulated expressions of VEGF and Ang-1 and promoted the formation of microvessels in brain tissues after TBI. Our study demonstrated the important role of BMSCs transplantation in TBI mouse and indicated that the underlying mechanism was through promoting angiogenesis and improving neurological function. This provides a novel and effective strategy for cell-based therapy in the treatment of TBI.

Establishment of an induced pluripotent stem cell line ZZUi003-A from a 65-year-old male with sporadic Parkinson's disease.

Skin fibroblasts were collected from a 65-year-old male patient with sporadic Parkinson's disease. Induced pluripotent stem cells were reprogrammed with human reprogramming factors (KMOSL) using the messenger RNA reprogramming protocol. The transgene-free iPSC line showed pluripotency, displayed normal karyotype, and could form embryoid bodies in vitro and differentiate into all 3 germ layers in vivo. This iPSC line can be a useful cellular model for studying the mechanism of Parkinson's disease.

Phosphorylation of c-Jun N-terminal kinase isoforms and their different roles in spinal cord dorsal horn and primary somatosensory cortex.

The present study was undertaken to investigate whether isoforms of c-Jun N-terminal kinase (JNK 46 kDa and 54 kDa), one component of the mitogen-activated protein kinase (MAPK) family, might show region-related differential activation patterns in both naïve and pain-experiencing rats. In naïve rats, no significant difference was observed in total expression level of the two JNK isoforms between spinal cord and primary somatosensory cortex (S1 area). However, phosphorylated JNK 46 kDa was normally expressed in the S1 area, but not in the spinal cord, while neither of the two structures contained phosphorylated JNK 54 kDa. Subcutaneous bee venom (BV)-induced persistent pain stimulation resulted in a significant increase in the phosphorylation of both JNK isoforms in each area for a long period (lasting at least 48 h). Nevertheless, JNK 46 kDa exhibited a much higher activation than JNK 54 kDa in the spinal cord, whereas the same noxious stimulation elicited evident activation of JNK 54 kDa in the S1 area, leaving JNK 46 kDa less affected. Intraplantar injection of sterile saline solution, causing acute and transient pain, produced almost the same changes in activation profile of the two JNK isoforms as found in the BV-treated rats. These results implicate that individual members of the JNK family may be associated with specific regions of nociceptive processing. Also, the two JNK isoforms are supposed to function differently according to their locations within the rat central nervous system.

Region- or state-related differences in expression and activation of extracellular signal-regulated kinases (ERKs) in naïve and pain-experiencing rats.

BACKGROUND: Extracellular signal-regulated kinase (ERK), one member of the mitogen-activated protein kinase (MAPK) family, has been suggested to regulate a diverse array of cellular functions, including cell growth, differentiation, survival, as well as neuronal plasticity. Recent evidence indicates a role for ERKs in nociceptive processing in both dorsal root ganglion and spinal cord. However, little literature has been reported to examine the differential distribution and activation of ERK isoforms, ERK1 and ERK2, at different levels of pain-related pathways under both normal and pain states. In the present study, quantitative blot immunolabeling technique was used to determine the spatial and temporal expression of ERK1 and ERK2, as well as their activated forms, in the spinal cord, primary somatosensory cortex (SI area of cortex), and hippocampus under normal, transient pain and persistent pain states. RESULTS: In naïve rats, we detected regional differences in total expression of ERK1 and ERK2 across different areas. In the spinal cord, ERK1 was expressed more abundantly than ERK2, while in the SI area of cortex and hippocampus, there was a larger amount of ERK2 than ERK1. Moreover, phosphorylated ERK2 (pERK2), not phosphorylated ERK1 (pERK1), was normally expressed with a high level in the SI area and hippocampus, but both pERK1 and pERK2 were barely detectable in normal spinal cord. Intraplantar saline or bee venom injection, mimicking transient or persistent pain respectively, can equally initiate an intense and long-lasting activation of ERKs in all three areas examined. However, isoform-dependent differences existed among these areas, that is, pERK2 exhibited stronger response than pERK1 in the spinal cord, whereas ERK1 was more remarkably activated than ERK2 in the S1 area and hippocampus. CONCLUSION: Taken these results together, we conclude that: (1) under normal state, while ERK immunoreactivity is broadly distributed in the rat central nervous system in general, the relative abundance of ERK1 and ERK2 differs greatly among specific regions; (2) under pain state, either ERK1 or ERK2 can be effectively phosphorylated with a long-term duration by both transient and persistent pain, but their response patterns differ from each other across distinct regions; (3) The long-lasting ERKs activation induced by bee venom injection is highly correlated with our previous behavioral, electrophysiological, morphological and pharmacological observations, lending further support to the functional importance of ERKs-mediated signaling pathways in the processing of negative consequences of pain associated with sensory, emotional and cognitive dimensions.