Chun Luo

Prognostic Influence and Magnetic Resonance Imaging Findings in Paroxysmal Sympathetic Hyperactivity after Severe Traumatic Brain Injury

Paroxysmal sympathetic hyperactivity (PSH) is a clinical syndrome affecting a subgroup of survivors of severe brain injury. In this study, the prevalence, magnetic resonance imaging (MRI) presentation, influence on the clinical course in the intensive care unit (ICU), and effect on neurological recovery of PSH were prospectively surveyed in 87 patients with severe traumatic brain injury (TBI). Cranial MRI was performed during the first 30 days after injury. The outcome was assessed according to the Glasgow Outcome Scale (GOS). PSH occurred in 18.4% of patients, with a greater incidence among younger patients and those with lower Glasgow Coma Scale (GCS) scores. Patients with PSH had more deep lesions as shown on cranial MRI, significantly longer ICU stays, and worse outcomes. PSH was shown to be common among patients with severe TBI who also had deep intraparenchymal lesions. The mechanism by which PSH influences patient outcomes has yet to be defined, but we believe that it may be mediated by diencephalic-mesencephalic dysfunction or disconnection.

RLIP76 is overexpressed in human glioblastomas and is required for proliferation, tumorigenesis and suppression of apoptosis

The guanosine triphosphatase-activating protein RLIP76 is overexpressed in many malignant tumor cells, but it is unclear if RLIP76 overexpression contributes to the high proliferative potential of glioma cells. We demonstrate that RLIP76 messenger RNA and protein expression are positively correlated with glioma grade and that higher RLIP76 expression correlates with shorter patient survival. Immunohistochemical staining revealed that RLIP76 expression was positively correlated with the expression of Ki-67, a biomarker for cell proliferation. Inhibition of RLIP76 expression in U87 and U251 glioma cell lines by stable transfection of a targeted siRNA suppressed anchorage-independent growth and enhanced apoptosis in vitro. Conversely, overexpression of RLIP76 in SW1088 and U251 cell lines enhanced proliferation and reduced apoptosis. Inhibition of RLIP76 in U251 cells also significantly suppressed tumorigenicity and induced apoptosis in an endotopic xenograft mouse model. Moreover, we demonstrate that knockdown of RLIP76 increases apoptosis in different human gliomas independently of p53 status. In addition, a constitutively active Rac1 reversed both the suppression of proliferation and the promotion of apoptosis induced by the RLIP76-targeted siRNA, indicating that RLIP76 is an upstream activator of Rac1. Rac1-mediated suppression of apoptosis and promotion of proliferation were dependent on intact c-jun N-terminal kinase (JNK) signaling. Furthermore, we demonstrate that RLIP76 promotes proliferation and suppresses glioma cell apoptosis through a mechanism independent of Rho-selective GTPase-activating protein. Instead, we found that the adenosine triphosphatase function of Rlip76 modulates Rac1 activity by regulating Rac1 protein ubiquitylation and degradation. These data demonstrate that RLIP76 may suppress apoptosis and promote the proliferation of glioma cells by direct adenosine triphosphate-dependent xenobiotic transport and by activating the Rac1-JNK signaling pathway. Inhibition of RLIP76 signaling is a potential treatment for malignant glioma.

Expression of ubiquitin-conjugating enzyme E2C/UbcH10 in astrocytic tumors.

UbcH10 is one of the key regulators of cell cycle progression through the mitotic spindle assembly checkpoint pathway. Recently, aberrantly high UbcH10 expression has been demonstrated in a variety of malignancies. However, its role in astrocytic carcinogenesis is not well defined. This study investigated the splice pattern of the UbcH10 gene and its expression status in astrocytomas of different grades. Consequently, UbcH10 splice variant 1 (GenBank accession nos. NM_007019) was detected in astrocytomas and normal brain tissues by RT-PCR and sequence analysis. Expression levels of UbcH10 mRNA were elevated in high- versus low-grade astrocytomas (64.33+/-60.98 vs 8.36+/-8.15, respectively; p=0.000) or normal controls (64.33+/-60.98 vs 1.00+/-1.57, respectively; p=0.000), as determined by quantitative real time PCR analysis. Similarly, immunohistochemistry study showed increased UbcH10 labelling index in high-grade astrocytomas versus low-grade tumors (10.53+/-5.79% vs 4.23+/-2.85%, respectively; p=0.000) or normal controls (10.53+/-5.79% vs 0.0+/-0.0%, respectively; p=0.000) and, a positive correlation between UbcH10 immunoreactivity and Ki-67 immunostaining was also noted (Spearman r=0.63, p<0.001). These data suggest that overexpression of UbcH10 may serve as one important molecular mechanism that underlies the astrocytic carcinogenesis.

Up-regulation of USP2a and FASN in gliomas correlates strongly with glioma grade

Gliomas are the most common neoplasms in the central nervous system. The lack of efficacy of glioma therapies necessitates in-depth studies of glioma pathology, especially of the underlying molecular mechanisms that transform normal glial cells into tumor cells. Here we report that a deubiquitinating enzyme, ubiquitin-specific protease 2a (USP2a), and its substrate, fatty acid synthase (FASN), are over-expressed in glioma tissue. Using real-time quantitative polymerase chain reaction (PCR), Western blot and immunohistochemistry, we examined the expression and cellular distribution of USP2a and FASN in human glioma tissues. The expression patterns of USP2a and FASN correlated with the pathologic and clinical characteristics of the patients. Real-time PCR analysis showed that the expression levels of USP2a and its substrate FASN were higher in high-grade (World Health Organization [WHO] grades III and IV) glioma tissues than in low-grade (WHO grades I and II) glioma tissues. Western blot analysis indicated that the average optical densitometry ratio of USP2a and its substrate FASN in high-grade gliomas was higher than in low-grade gliomas. Moreover, statistical analysis of grade-classified glioma samples showed that the level of USP2a and FASN expression increased with the elevation of the WHO grade of glioma. USP2a protein expression was detected in the nucleus of glioma tissues and an increase in expression was significantly associated with the elevation of the WHO grade of glioma by immunohistochemistry. These findings expand our understanding of the molecular profiling of glioma and could shed light on new diagnostic criteria for gliomas.

Knockdown of ubiquitin-conjugating enzyme E2C/UbcH10 expression by RNA interference inhibits glioma cell proliferation and enhances cell apoptosis in vitro

PurposeTo address the role of ubiquitin-conjugating enzyme, E2C/UbcH10, in astrocytic carcinogenesis.MethodsExpression pattern of UbcH10 in U251 glioma cells was evaluated by immunohistochemistry and western blot. RNA interference was employed to downregulate UbcH10 expression in U251 cell line. The effect of UbcH10 silencing on cell proliferation was assessed by MTT assay and cell cycle analysis. Cell apoptosis was determined by flow cytometry, TUNEL staining and western blot.ResultsLevels of UbcH10 protein were significantly upregulated in U251 cells compared with normal brain tissues. Marked immunoreactivity for UbcH10 was demonstrated in the cytoplasm of U251 glioma cells, especially in the mitotic cells. The growth rate of U251 cells was significantly inhibited by depletion of UbcH10 by short interference RNA. Further, UbcH10 RNAi induced apoptosis through induction of Bax and p53, downregulation of Bcl-2 and G2/M arrest of the cell cycle.ConclusionThese data imply that knocking-down UbcH10 protein expression may represent a potential therapeutic option for glioma.

Knockdown of AKT2 expression by RNA interference inhibits proliferation, enhances apoptosis, and increases chemosensitivity to the anticancer drug VM-26 in U87 glioma cells

The AKT2 kinase (protein kinas Bβ) is frequently overexpressed in malignant gliomas. In this study, the human glioblastoma cell line U87 was stably transfected with a lentivirus vector expressing a short hairpin RNA (shRNA) targeting AKT2. Knockdown of AKT2 by the shRNA inhibited U87 cell proliferation and increased the rate of apoptosis. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot analysis revealed that cells stably underexpressing AKT2 showed lower expression of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) and enhanced expression of the apoptosis effector caspase-3 compared to U87 cells stably transfected with a control vector. Furthermore, expression levels of AKT2 were correlated with the IC50 of the antitumor drug VM-26 (teniposide); the VM-26 IC50 was reduced from 6.46±0.42μg/ml in control glioma cells to 1.15±0.22μg/ml in U87 cells underexpressing AKT2. Combined AKT2 knockdown and VM-26 treatment inhibited cell proliferation in vitro more effectively than either treatment alone. Knockdown of AKT2 expression was associated with decreased expression of the multidrug resistance-associated protein 1 (MRP1) without affecting MRP1 mRNA expression. However, the mRNA and protein levels of MDR1 (p-glycoprotein) were unaffected by AKT2 knockdown. These results indicate that inhibition of AKT2 expression may be an effective means for overcoming AKT2-associated chemoresistance in human malignant glioma cells and may represent a potential gene-targeting approach to treat glioma.

Expression of targeting protein for Xenopus kinesin-like protein 2 is associated with progression of human malignant astrocytoma.

In humans, the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cell cycle-associated protein, and altered TPX2 expression has been found in various malignancies. However, the contribution of TPX2 expression to astrocytoma progression is unclear. The aim of this study was to investigate TPX2 expression in human astrocytoma samples and cell lines. TPX2 protein expression was detected in the nucleus of astrocytoma tissues by immunohistochemistry and immunofluorescence staining. Real-time PCR and Western blot analysis showed that the expression levels of TPX2 were higher in high-grade astrocytoma tissues and cell lines than that in low-grade astrocytoma tissues and normal cell lines. Immunohistochemical analysis of tumor tissues from 52 patients with astrocytoma showed that TPX2 over-expression was significantly associated with decreased patient survival. In addition, down-regulation of the TPX2 gene by RNA interference inhibited proliferation of U87 cells. TPX2 gene silencing also increased early-stage apoptosis in U87 cells. Western blotting and real-time PCR showed changes in the protein and mRNA expression of Aurora A, Ran, p53, c-Myc and cyclin B1 in U87 cells that had been transfected with pSUPER/TPX2/siRNA. These data suggest that TPX2 expression is associated with the progression of malignant astrocytoma.

Risk factors related to dysautonomia after severe traumatic brain injury.

BACKGROUND Dysautonomia after severe traumatic brain injury (TBI) is a clinical syndrome affecting a subgroup of survivors and is characterized by episodes of autonomic dysregulation and muscle overactivity. The purpose of this study was to determine the incidence of dysautonomia after severe TBI in an intensive care unit setting and analyze the risk factors for developing dysautonomia. METHODS A consecutive series of 101 patients with severe TBI admitted in a major trauma hospital during a 2-year period were prospectively observed to determine the effects of age, sex, mode of injury, hypertension history, admission systolic blood pressure, fracture, lung injury, admission Glasgow Coma Scale (GCS) score, injury severity score, emergency craniotomy, sedation or analgesia, diffuse axonal injury (DAI), magnetic resonance imaging (MRI) scales, and hydrocephalus on the development of dysautonomia. Risk factors for dysautonomia were evaluated by using logistic regression analysis. RESULTS Seventy-nine of the 101 patients met inclusion criteria, and dysautonomia was observed in 16 (20.3%) of these patients. Univariate analysis revealed significant correlations between the occurrence of dysautonomia and patient age, admission GCS score, DAI, MRI scales, and hydrocephalus. Sex, mode of injury, hypertension history, admission systolic blood pressure, fracture, lung injury, injury severity score, sedation or analgesia, and emergency craniotomy did not influence the development of dysautonomia. Multivariate logistic regression revealed that patient age and DAI were two independent predictors of dysautonomia. There was no independent association between dysautonomia and admission GCS score, MRI scales, or hydrocephalus. CONCLUSIONS Dysautonomia frequently occurs in patients with severe TBI. A younger age and DAI could be risk factors for facilitating the development of dysautonomia.

Knockdown of CDK6 enhances glioma sensitivity to chemotherapy

Chemotherapy is widely used for the treatment of glioma. Given the high resistance of brain neoplasm tissues to chemotherapy, it is important to find new methods to improve the effects of chemotherapy. However, the molecular mechanisms underlying glioma resistance to chemotherapy are largely unknown. Here, we demonstrate that CDK6, a cell cycle regulator, is significantly upregulated in glioma cells, and the increasing expression of CDK6 correlates well with the grades of glioma malignancy. Using shRNA-mediated CDK6 knockdown, we found that the proliferation and survival of tumor cells were dramatically inhibited. Moreover, CDK6 knockdown in the U251 glioma cell line caused significant increase in the apoptosis of U251 cells treated with temozolomide (TMZ). Furthermore, CDK6 knockdown reduced the expression level of drug resistance genes such as MRP and MDR. These data indicate that CDK6 is an important mediator of glioma resistance to chemotherapy. Our findings provide a new strategy for the development of chemotherapy sensitizer.

Differential Expression of Circular RNAs in Glioblastoma Multiforme and Its Correlation with Prognosis

OBJECTIVE: The present study aimed to explore the expression profiles of circular RNAs (circRNAs) in glioblastoma multiforme (GBM) in an attempt to identify potential core genes in the pathogenesis of this tumor. METHODS: Differentially expressed circRNAs were screened between tumor tissues from five GBM patients and five normal brain samples using Illumina Hiseq. Bioinformatics analysis was used to analyze their potential function. CircBRAF was further detected in different WHO grades glioma tissues and normal brain tissues. Kaplan-Meier curves and multivariate Coxs analysis were used to analyze the association between circBRAF expression level and prognosis of glioma patients. RESULTS: A total of 1411 differentially expressed circRNAs were identified in GBM patients including 206 upregulated circRNAs and 1205 downregulated circRNAs. Differential expression of circRNAs was closely associated with the biological process and molecular function. The downregulated circRNAs were mainly associated with ErbB and Neurotrophin signaling pathways. Moreover, the expression level of circBRAF in normal brain tissues was significantly higher than that in glioma tissues (P < .001). CircBRAF was significantly lower in glioma patients with high pathological grade (WHO III & IV) than those with low grade (WHO I & II) (P < .001). Cox analysis revealed that high circBRAF expression was an independent biomarker for predicting good progression-free survival and overall survival in glioma patients (HR = 0.413, 95% CI 0.201-0.849; HR = 0.299, 95% CI 0.135-0.661; respectively). CONCLUSION: The present study identified a profile of dysregulated circRNAs in GBM. Bioinformatics analysis showed that dysregulated circRNAs might be associated with tumorigenesis and development of GBM. In addition, circBRAF could severe as a biomarker for predicting pathological grade and prognosis in glioma patients.