LncRNA NEAT1 promotes malignant phenotypes and TMZ resistance in glioblastoma stem cells by regulating let-7g-5p/MAP3K1 axis.

Glioblastoma multiforme (GBM) is one of the most malign brain tumors in adults. Temozolomide (TMZ) is an oral chemotherapy drug constituting the backbone of chemotherapy regimens utilized as first-line treatment of GBM. However, resistance to TMZ often leads to treatment failure. In the present study, we explored the expression and related mechanisms of nuclear enriched abundant transcript 1 (NEAT1) in glioma stem cells (GSCs). Quantitative real-time PCR (qRT-PCR) showed that NEAT1 was up-regulated in serum samples of GBM patients and GSCs isolated from U87, U251 cell lines. Functional experiments showed that NEAT1 knockdown restrained malignant behaviors of GSC, including proliferation, migration and invasion. Dual-luciferase assays identified let-7g-5p was a downstream target and negatively adjusted by NEAT1. Restoration of let-7g-5p impeded tumor progression by inhibiting proliferation, migration and invasion. Mitogen-activated protein kinase kinase kinase 1 (MAP3K1), as a direct target of let-7g-5p, was positively regulated by NEAT1 and involved to affect the regulation of NEAT1 on GSCs' behaviors. In conclusion, our results suggested that NEAT1 promoted GSCs progression via NEAT1/let-7g-5p/MAP3K1 axis, which provided a depth insight into TMZ resistance mechanism.

Different expression of miR-29b and VEGFA in glioma.

Glioma is one of the most common carcinomas in terms of both incidence and mortality worldwide. This is a case-control study with 240 cases and age- and gender-matched controls in the rate of 1:1. The results of this present word indicated that the expressions of both miR-29b and VEGFA in blood were significantly different compared with the control group, and thus may help to differentiate glioma cases from the controls. In addition, the diagnostic role of miR-29b and VEGFA was important for the clinical application. In conclusion, circulating miR-29b and VEGFA could be used as diagnosis biomarkers.

Decreased expression of the SPOP gene is associated with poor prognosis in glioma.

This study suggests that speckle-type POZ protein (SPOP) may be a tumor suppressor gene and its prognostic value in human glioma. Real-time quantitative RT-PCR (qRT­PCR), western blotting, and immunohistochemical staining were used to examine SPOP expression in glioma tissues and normal brain (NB) tissues. The relationships between the SPOP expression levels, the clinicopathological factors, and patient survival were investigated. The molecular mechanisms of SPOP expression and its effects on cell viability, migration and invasion were also explored by MTT assay, wound-healing assays and Transwell assay. SPOP mRNA and protein levels were downregulated in glioma tissues compared to NB. Immunohistochemical staining results showed low expression in 62.2% (61/98) of glioma samples, while high expression in 75% (9/12) of NB samples, and the difference was statistically significant (P=0.014). In addition, decreased SPOP was associated disease progression in glioma samples, the expression level of SPOP was positively correlated with mean tumor diameter (MTD) (P=0.021) and the status of tumor grade and histological type (WHO I, II, III and IV) (P=0.032) in glioma patients. Additionally, the overall survival of patients with low SPOP expression was significantly worse than that of SPOP-high patients (P=0.001). In vitro overexpression of SPOP markedly inhibited cell viability, migration and invasion in vitro. These findings suggest that SPOP has potential use as novel biomarker of glioma and may serve as an independent predictive factor for prognosis of glioma patients.

miR-155 Regulates Glioma Cells Invasion and Chemosensitivity by p38 Isforms In Vitro.

The critical role of microRNAs in cancer development has been extensively described. miRNAs are both specific markers and putative therapy targets. miR-155 has been identified to be an oncomiRNA and is highly expressed in several solid cancers, including glioblastoma. In this study, we found that miR-155 is a good potential therapy target. Knockdown of miR-155 sensitizes glioma cells to the chemotherapy of temozolomide (TMZ) by targeting the p38 isoforms mitogen-activated protein kinase 13 [MAPK13, also known as p38 MAPKδ or stress-activated protein kinase 4 (SAPK4)] and MAPK14 (also known as p38 MAPKα). As tumor suppressor genes, MAPK13 and MAPK14 play important roles in lowering the accumulation of reactive oxygen species (ROS), inducing cell apoptosis, and slowing the progression of cancer. Knockdown of miR-155 enhanced the anticancer effect of TMZ on glioma by targeting the MAPK13 and MAPK14-mediated oxidative stress and apoptosis, but did not affect the secretion of MMP2 and MMP9.

Comparison of administration routes for adipose-derived stem cells in the treatment of middle cerebral artery occlusion in rats.

Given that adult adipose tissue is an abundant, accessible and safe source of stem cells, the use of adipose-derived stem cells (ADSCs) provides a promising approach in ischemic stroke. The delivery route, however, for transplantation of ADSCs in clinical application remains controversial regarding the time window, cell type, safety issues, 'first pass' effect and therapeutic effect. To determine the optimal administration route in transplantation of ADSCs, we compared the therapeutic effect of the three mainly used administration routes of ADSCs in a middle cerebral artery occlusion (MCAO) rat model. Cells isolated from the adipose tissue of adult rodents were differentiated and characterized in vitro, and further transplanted in vivo by intravenous, intra-arterial or intra-ventricular delivery. The infarct volume, expression of neurotrophic factors and the neurobehavioral improvements were evaluated after the equal dose of BrdU labeled ADSCs transplantation. Our results indicated that the equal dose of ADSCs delivered intravenously were effective in improving the neurological outcome and reducing the infarct volume after ischemic brain injury in long term duration in contrast to intra-arterial and intra-ventricular delivery. At 1-7 days after transplantation, the increased expression levels of BDNF, VEGF, bFGF, Bcl-2, IL-10 and decreased levels of caspase-3 and TNF-α in the intra-ventricular and intra-arterial groups were significant in contrast to the intravenous group. There was no significant difference among the three groups after 7 days. Our findings suggest that compared with the intra-ventricular delivery, intravascular injection allows higher dose injection with fewer invasions and appears to be optimal in application with regard to therapeutic efficacy, safety and feasibility.

Schwann cells differentiated from adipose­derived stem cells for the treatment of brain contusion.

Rapid development of tissue engineering techniques has led to the possibility of treating central nervous injuries with Schwann cells (SCs). However, certain characteristics of SCs, such as a low proliferation ability, greatly restrict their use. The aim of the present study was to investigate whether SCs differentiated from adipose­derived stem cells (ADSC­SCs) could used to promote functional recovery in brain contusion in rat. ADSCs were isolated and expanded from the groin of Sprague­Dawley rats and differentiated into SCs. The ADSC­SCs were transplanted into the contused rat brain and the locomotor function of the rats was assessed. Significant locomotor function recovery was observed in hemiparalyzed rats treated with ADSCs­SCs. In conclusion, transplantation of ADSC­SCs significantly promoted functional recovery following brain contusion.

Role of podocalyxin in astrocytoma: Clinicopathological and in vitro evidence.

The present study examined the expression of podocalyxin (PODX) in surgically-resected astrocytomas, associated the levels of PODX expression with the clinicopathological characteristics and survival outcomes of astrocytoma and assessed how PODX affected the viability of astrocytoma cells following the administration of chemotherapeutic agents. The immunohistochemical analysis of 102 patient samples revealed that a high expression of PODX was significantly associated with high-grade astrocytomas (P<0.001) and a high Ki-67 labeling index (LI; P<0.001). A Kaplan-Meier survival analysis demonstrated that the high PODX expression group had significantly shorter disease-free survival (DFS) and overall survival (OS) rates compared with the low expression group (P<0.001). The multivariate analysis using the Cox's proportional hazards model revealed that a high expression of PODX, a high World Health Organization grade and a high Ki-67 LI were independent factors for shorter DFS and OS times. A subsequent in vitro study using SW1783 and U-87 human astrocytoma cell lines revealed that knocking down PODX decreased astrocytoma cell viability against temozolomide-induced apoptotic stress through the inhibition of the Akt survival signaling pathway. In conclusion, the in vivo findings indicated that a high expression of PODX is predictive of a poor survival outcome and, thus, may be used as a prognostic factor to predict the survival outcomes of astrocytoma patients. The in vitro findings indicated that PODX may promote astrocytoma cell viability against chemotherapeutic agent-induced apoptotic stress through the Akt pathway, indicating that PODX may be a novel target for overcoming chemoresistance in astrocytomas.

Let-7b expression determines response to chemotherapy through the regulation of cyclin D1 in glioblastoma.

BACKGROUND: Glioblastoma is the most common type of primary brain tumors. Cisplatin is a commonly used chemotherapeutic agent for Glioblastoma patients. Despite a consistent rate of initial responses, cisplatin treatment often develops chemoresistance, leading to therapeutic failure. Cellular resistance to cisplatin is of great concern and understanding the molecular mechanisms is an utter need. METHODS: Glioblastoma cell line U251 cells were exposed to increasing doses of cisplatin for 6 months to establish cisplatin-resistant cell line U251R. The differential miRNA expression profiles in U251 and U251R cell lines were identified by microarray analysis and confirmed by Q-PCR. MiRNA mimics were transfected into U251R cells, and cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. RESULTS: U251R cells showed 3.1-fold increase in cisplatin resistance compared to its parental U251 cells. Microarray analysis identified Let-7b and other miRNAs significantly down-regulated in U251R cells compared to U251 cells. Transfection of Let-7b mimics greatly re-sensitized U251R cells to cisplatin, while transfection of other miRNAs has no effect or slightly effect. Cyclin D1 is predicted as a target of Let-7b through bioinformatics analysis. Over-expression of Let-7b mimics suppressed cyclin D1 protein expression and inhibited cyclin D1-3'-UTR luciferase activity. Knockdown of cyclin D1 expression significantly increased cisplatin-induced G1 arrest and apoptosis. CONCLUSIONS: Collectively, our results indicated that cisplatin treatment leads to Let-7b suppression, which in turn up-regulates cyclin D1 expression. Let-7b may serve as a marker of cisplatin resistance, and can enhance the therapeutic benefit of cisplatin in glioblastoma cells.

Podocalyxin regulates astrocytoma cell invasion and survival against temozolomide.

Increased podocalyxin (PODXL) expression has been associated with a subset of aggressive types of cancer. To the best of our knowledge, the effect of PODXL on astrocytoma cell invasion and survival against chemotherapy agent was investigated for the first time in the present study. Overexpression and knockdown of PODXL were respectively performed in SW1783 (grade III astrocytoma) and U-87 (grade IV astrocytoma; gliobalstoma) cells. PODXL overexpression in SW1783 cells significantly increased cell invasion, matrix metalloproteinase-9 (MMP-9) expression, cell survival against temozolomide-induced apoptotic stress, and phosphorylation of Akt at serine 473 (ser473), which was abolished by the selective phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY). Knockdown of PODXL in U-87 cells significantly decreased cell invasion, MMP-9 expression, cell survival against temozolomide, and phosphorylation of Akt at serine 473 (ser473), which was further decreased by LY treatment. In conclusion, in the present study it was demonstrated that PODXL promotes astrocytoma cell invasion, potentially through the upregulation of MMP-9 expression in a PI3K-dependent manner. Additionally, PODXL was shown to promote astrocytoma cell survival against temozolomide-induced apoptotic stress by enhancing the activation of the PI3K/Akt survival signaling pathway. This study provides novel insights into the molecular mechanisms underlying astrocytoma progression, cell survival and chemoresistance, and suggests that PODXL may be a potential target for overcoming chemoresistance in astrocytomas.

Circulating miR-17, miR-20a, miR-29c, and miR-223 combined as non-invasive biomarkers in nasopharyngeal carcinoma.

BACKGROUND: MicroRNAs have been considered as a kind of potential novel biomarker for cancer detection due to their remarkable stability in the blood and the characteristics of their expression profile in many diseases. METHODS: We performed microarray-based serum miRNA profiling on the serum of twenty nasopharyngeal carcinoma patients at diagnosis along with 20 non-cancerous individuals as controls. This was followed by a real-time quantitative Polymerase Chain Reaction (RT-qPCR) in a separate cohort of thirty patients with nasopharyngeal carcinoma and thirty age- matched non-cancerous volunteers. A model for diagnosis was established by a conversion of mathematical calculation formula which has been validated by analyzing 74 cases of patients with nasopharyngeal carcinoma and 57 cases of non-cancerous volunteers. RESULTS: The profiles showed that 39 and 17 miRNAs are exclusively expressed in the serum of non-cancerous volunteers and of patients with nasopharyngeal carcinoma respectively. 4 miRNAs including miR-17, miR-20a, miR-29c, and miR-223 were found to be expressed differentially in the serum of NPC compared with that of non-cancerous control. Based on this, a diagnosis equation with Ct difference method has been established to distinguish NPC cases and non-cancerous controls and validated with high sensitivity and specificity. CONCLUSIONS: We demonstrate that the serum miRNA-based biomarker model become a novel tool for NPC detection. The circulating 4-miRNA-based method may provide a novel strategy for NPC diagnosis.

Wnt inhibitory factor-1 regulates glioblastoma cell cycle and proliferation.

Wnt proteins are powerful regulators of cell proliferation and differentiation, and activation of the Wnt signalling pathway is involved in the pathogenesis of several types of human tumours. Wnt inhibitory factor-1 (WIF-1) acts as a Wnt antagonist and tumour suppressor. Previous studies have shown that reducing expression of the WIF-1 gene aberrantly activates Wnt signalling and induces the development of certain types of cancers. In the present study, we examined the expression of WIF-1 in human primary glioblastoma multiforme (GBM) tumours. Studies using semiquantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis revealed that WIF-1 expression is lower in human GBM than in normal brain tissue. To clarify the role of WIF-1, we transfected U251 human glioblastoma-derived cells, which do not express WIF-1, with the pcDNA3.1-WIF1 vector to restore WIF-1 expression. The results of cell proliferation, colony formation and apoptosis assays, as well as flow cytometry, indicate that exogenous WIF-1 has no effect on U251 cell apoptosis, but does arrest cells at the G(0)/G(1) phase and inhibit cell growth. Collectively, our data suggest that WIF-1 is a potent inhibitor of GBM growth.

RRP22: a novel neural tumor suppressor for astrocytoma.

Astrocytomas are the most common neoplasm of the central nervous system. Although progress has been made, the survival rate of astrocytoma is still poor. Therefore, improving the prognosis of patients with astrocytomas relies on effective therapies that are directed against unique molecular aberrations. Previous studies have revealed that a novel member of the Ras superfamily, RRP22, which is located on chromosome 22 on the 12q site, is exclusively expressed in the central nervous system. RRP22 can be modified by farnesyl and down-regulated in a variety of neural tumor cell lines. In this study, we analyzed the mRNA level of RRP22 in normal brain tissues and astrocytomas using quantitative RT-PCR. Our results showed that the mRNA level in astrocytomas was significantly down-regulated compared to levels in normal tissues. As the pathological grade (World Health Organization (WHO) classification 2007) increased, the expression of RRP22 decreased. However, according to our research, there was no significant difference between malignant astrocytomas with pathological grades of III or IV. To investigate the possible effects of RRP22 on the biological behavior of glioma cells, we transfected RRP22 into a malignant cell line of astrocytomas, U251. We found that RRP22 inhibited growth, decreased invasiveness, and induced cell death. Thus, RRP22 is a special neural tumor suppressor for human astrocytomas, although further studies are needed to define the detailed mechanisms.

Expression of TNKS1 is correlated with pathologic grade and Wnt/ß-catenin pathway in human astrocytomas.

Astrocytoma is the most common neoplasm of the central nervous system, and its malignancy is closely related to activation of the Wnt/ß-catenin pathway. Accumulated evidence shows that tankyrase (TNKS) is necessary for the Wnt/ß-catenin pathway, stabilizing ß-catenin, and that TNKS1, a major member of the TNKS family, is involved in a wide range of human cancers. However, the expression of TNKS1 and the molecular relationship between TNKS1 and ß-catenin in human astrocytomas is largely unknown. In the present study, we investigated the expression of TNKS1 in human astrocytomas using reverse transcription-polymerase chain reaction, Western blot and immunohistochemistry. The mRNA and protein expression levels of TNKS1 in astrocytomas were significantly higher compared with the normal brain tissues. Significant association between TNKS1 upregulation and pathological grade of astrocytomas was also confirmed. In addition, ß-catenin immunostaining of the sampled tissues revealed a highly similar change to TNKS1. This study provides additional evidence for the involvement of TNKS1 gene and the Wnt/ß-catenin signaling pathway in the genesis and progression of astrocytoma. TNKS1 may have a key role in astrocytomas.

Downregulation of WIF-1 by hypermethylation in astrocytomas.

Wnt inhibitory factor-1 (WIF-1) acts as a Wnt antagonist and tumor suppressor, but hypermethylation of WIF-1 gene promoter and low expression of WIF-1 activate Wnt signaling aberrantly and induce the development of several human tumors. By using RT-PCR, immunohistochemistry and methylation-specific PCR, we analyzed the expression and methylation of WIF-1 in 4 normal brain tissues, 35 freshly resected astrocytoma tissues and 4 glioblastoma-derived cell lines. Significant downregulation of WIF-1 mRNA and protein expression levels was observed in astrocytoma tissues compared with normal brain tissues. Significant association between WIF-1 downregulation and pathological grade of astrocytomas was found. WIF-1 gene aberrant methylation was observed in 19 of 35 (54.29%) tumor samples. The promoter methylation tumors showed low WIF-1 protein and mRNA expression, whereas the promoter unmethylation tumors displayed high protein and mRNA expression levels. Moreover, complete absence of WIF-1 mRNA expression was observed in four cell lines, whereas treatment with demethylating agent, 5-aza-2'-deoxycytidine, restored WIF-1 expression. Our results suggested that the WIF-1 gene is frequently silenced in astrocytoma by aberrant promoter methylation. This may be an important mechanism in astrocytoma carcinogenesis.

Expression and aberrant promoter methylation of Wnt inhibitory factor-1 in human astrocytomas.

BACKGROUND: Wnt inhibitory factor-1(WIF-1) acts as a Wnt-antagonists and tumor suppressor, but hypermethylation of WIF-1 gene promoter and low expression activate Wnt signaling aberrantly and induce the development of various human tumors. With this work we intended to investigate the expression and promoter methylation status of WIF-1 gene in human astrocytomas. METHODS: The tissue samples consisted of 53 astrocytomas and 6 normal brain tissues. The expression levels of WIF-1 were determined by immunohistochemistry and semiquantitative RT-PCR. The results were analyzed in correlation with clinicopathological data. Methylation status of WIF-1 gene promoter was investigated using methylation specific PCR. The relationship between methylation and expression of the genes was analyzed. RESULTS: The average expression levels of WIF-1 protein and mRNA in astrocytomas were decreased significantly compared with normal control tissues. The protein and mRNA expression of WIF-1 gene in astrocytomas was decreased with the increase of pathological grade. Furthermore, WIF-1 promoter methylation was observed by MS-PCR in astrocytomas which showed significant reduction of WIF-1 expression. The WIF-1 promoter hypermethylation was associated with reduced expression of WIF-1 expression. CONCLUSION: Our results demonstrate that the WIF-1 gene is frequently down-regulated or silenced in astrocytomas by aberrant promoter methylation. This may be an important mechanism in astrocytoma carcinogenesis.

[Clinical significance of BMP-2 protein and mRNA expression in human glioma].

AIM: To investigate the expression of BMP-2 protein and mRNA in glioma, and evaluate its clinical significance in clinicopathological status of patients with glioma. METHODS: BMP-2 protein and mRNA expression in 88 patients with glioma were examined by SABC immunohistochemistry and RT-PCR, respectively. RESULTS: The positive expression rate of BMP-2 protein in glioma tissues were 78.40% (69/88), which was significantly higher (P<0.01) than that in normal brain tissues [3% (6/20)]. The expression of BMP-2 protein and mRNA in gliomas of grade III-IV was also significantly higher than that in gliomas of grade I-II. CONCLUSION: BMP-2 expression in cancerous tissues may play an important role in the progression of glioma, which may help to predict patients' prognosis as a tumor marker.

[Microsurgical treatment and prevention of postoperative complications for the fourth ventricle tumors in adults].

OBJECTIVE: To explore the microneurosurgical technique and prevention of postoperative complications for the fourth ventricle tumors in adults. METHODS: We retrospectively analyzed the clinical data of 68 patients with the fourth ventricle tumors between August 2005 and August 2007 in Xiangya Hospital after microsurgical operation. Tumors were excised by inferior vermis cerebellar approach or cerebellomedullary fissure approach. The extent of tumor removal should take into consideration the possible injury of brain stem respiratory center, especially tumors adherent to the brain stem. Cerebral aqueduct obstructions were removed in all patients, suspending dura on the neck muscles during closing skull to eliminate scalp hydrops. RESULTS: There were 58 total tumor excisions and 10 subtotal tumor excisions. No patient died and no suboccipital hydrops took place before discharge in this study. Postoperative symptomatic hydrocephalus was found in 10 patients, but it was cured by ventricle-abdomen shunt. Hemorrhage in tumor lumen happened in 4 patients, who received second microsurgery. Drugs were given to 8 patients with intracranial pneumatocele, 10 with intracranial infection, and 18 with upper gastrointestinal hemorrhage. Five patients out of the 16 tracheotomies recovered well by mechanical ventilation. CONCLUSION: Protecting the life center of brain stem and dredging the aqueduct outlet completely were the key to surgical success. Therapeutic effect could be improved by adept microneurosurgical techniques after operation. The prognosis of patients may be improved by preventing complications actively and combined therapy after the operation.

[Mutation analysis of neural cell adhesion molecules in human astrocytoma].

OBJECTIVE: To identify the function of neural cell adhesion molecule (NCAM) mutation in the genesis of human glioma. METHODS: Mutations were found through polymerase chain reaction-single strand conformation polymorphism, and then the changed DNA fragments were purified and multiplied and sent to Shanghai for sequencing. Blast and ORF finder were used to find out the amino changes in NCAM. RESULTS: An A-C transversion was found at position 1 126 in NCAM's 7 exon in a patient with glioblastoma from 43 astrocytoma. CONCLUSION: Structural change in the protein caused by point mutation may be the reason for tumorigenesis of astrocytoma.

[Microsurgery techniques for insular glioma].

OBJECTIVE: To explore the microsurgical techniques for insular glioma without damaging its surrounding normal structures. METHODS: We retrospectively analyzed 54 patients with insular gliomas who underwent microsurgical operation by trans-syvian fissure approach between May, 2003 and August, 2008 in Xiangya Hospital. We discussed the techniques in the operation and summarized how to protect the key blood vessels, distinguish and protect the surrounding normal structures. RESULTS: There were 36 complete removals,14 secondary complete removals, and 4 partial removals.Six patients had complications after the craniotomy who had temporal speech disorder (aphasia mostly began to recover about 10 days after the craniotomy),4 patients had opposite side paralysis worsening (3 recovered normally and 1 improved after 6 months),4 had light paralysis, and another 3 had paralysis and speech disorder. CONCLUSION: The microsurgery by means of trans-syvian fissure approach can well expose the anatomical relation between tumor and its surrounding structures,so that we can remove the tumor and protect the surrounding normal tissues as much as we can.

Vestibular schwannoma microsurgery with special reference to facial nerve preservation.

OBJECTIVE: To retrospectively study the outcomes of vestibular schwannoma (VS) resection. METHODS: Between January 2003 and December 2006, 103 consecutive patients who had undergone VS resection were included in this study. Medical records, operation summaries, follow-up data, and neuroradiological findings were analyzed. The relationship between tumor size, location, and topography relative to the facial nerve bundles was studied for a mean duration of 16 months (range: 3-39 months). RESULTS: Complete tumor resection in combination with anatomic preservation of the facial nerve was achieved in 101 (98.1%) cases. The facial nerve was fully preserved in 100% of cases with small or medium tumors and in 37/39 patients with large tumors. Overall, 83.5% of patients had normal or near-normal facial nerve function 3-12 months post-surgically. The mortality rate was 0%. CONCLUSIONS: Even in large VS, preservation of facial nerve function (H-B Grade I or II) should be prioritized over total resection. For tumors >3cm, the goal of low morbidity and maintenance of normal facial nerve function can be attained with the retrosigmoid transmeatal approach, refined microsurgical technique, and intraoperative facial nerve monitoring.