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.

[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.

[Electrocorticography monitoring in microsurgical treatment of solitary cavernous angiomas].

OBJECTIVE: To investigate the curative effect of electrocorticography (ECoG) monitoring in the microsurgical treatment of cavernous angiomas. METHODS: Clinical data of 71 patients with epileptogenic cavernous angiomas,who had been performed ECoG monitoring during the operation,were analyzed retrospectively. RESULTS: The foci of cavernous angiomas and epilepsy of the 71 patients were resected during the operation. In the 58 patients who were followed up,42 had not epileptic seizure,and 16 still had epileptic seizure,while the frequencies of 13 patients reduced to below 10%,and 3 patients over 10%. CONCLUSION: The drug treatment of epileptogenic cavernous angiomas can not control epileptic seizure,and the patients should receive the microsurgical treatment early. Electrocorticography monitoring can direct the surgical procedure,and control the postoperative epileptic seizure.

[Chemoresistance of CD133(+) tumor stem cells from human brain glioma].

OBJECTIVE: To explore the multidrug resistance (MDR) mechanism of ABC superfamily transporters in the tumor stem cells(TSC) from human brain glioma tissues. METHODS: Samples of glioma were obtained from 30 patients undergoing microsurgical tumor resection. The CD133(+) cells and CD133(-) cells from these tumor specimens were isolated by magnetic activated cell sorting(MACS). These cells were cultured, proliferated and passaged. The protein and activity expression of multidrug-resistance protein 1(MDR1) and multidrug-resistance associated protein 1(MRP1) were analyzed between CD133(+) and CD133(-) cells by immunocytochemistry and RT-PCR respectively. RESULTS: CD133(+) cells generated free floating neurosphere like brain tumor spheres(BTS) and abnormal proliferating capacity in the serum-free medium(SFM) in vitro. Three cases from glioblastoma stem cells could form BTS in the complete medium, and could be cultured for 1-3 passages. The range of positive cell proportion for MDR1 and MRP1 expression in CD133(+) cells was 18%-67% and 23%-73% respectively. The expression levels of MDR1 and MRP1 mRNA were higher in CD133(+) glioma stem cells than those in the differentiated tumor cells(TC), the protein activity was increased to 16.1 and 19.6 times respectively compared with that of TC. The protein and activity expression were positively related to the pathological grades of tumors. MDR1 or MRP1 drug resistance was not expressed in all the tumors and there was obvious correlation between MDR1 and MRP1. CONCLUSION: Only a small proportion of cells in the heterogeneous glioma is CD133(+) brain tumor stem cells which display the strong capacity of self-renewing, abnormal proliferation and intrinsic multidrug resistance to traditional chemotherapy. The high expression of MDR1 and MRP1 by the CD133(+) brain tumor stem cells is one of the main mechanisms in the chemoresistance of tumors. CD133(+) brain tumor stem cells can be served as the root of multidrug resistance and key therapeutic target for glioma chemotherapy.

[Isolation and identification of brain tumor stem cells from human brain neuroepithelial tumors].

OBJECTIVE: To establish a simplified culture system for the isolation of brain tumor stem cells (BTSCs) from the tumors of human neuroepithelial tissue, to observe the growth and differentiation pattern of BTSCs, and to investigate their expression of the specific markers. METHODS: Twenty-six patients with brain neuroepithelial tumors underwent tumor resection. Two pieces of tumor tissues were taken from each tumor to be dissociated, triturated into single cells in sterile DMEM-F12 medium, and then filtered. The tumor cells were seeded at a concentration of 200,000 viable cells per mL into serum-free DMEM-F12 medium simply supplemented with B27, human basic fibroblast growth factor (20 microg/L), human epidermal growth factor (20 microg /L), insulin (4 U/L), L-glutamine, penicillin and streptomycin. After the primary brain tumor spheres (BTSs) were generated, they were triturated again and passed in fresh medium. Limiting dilution assay was performed to observe the monoclone formation. 5-bromodeoxyuridine (BrdU) incorporation test was performed to observe the proliferation of the BTS. The BTSCs were cultured in mitogen-free DMEM-F12 medium supplemented with 10% fetal bovine serum to observe their differentiation. Immunocytochemistry was used to examine the expression of CD133 and nestin, specific markers of BTSC, and the rate of CD133 positive cells. RESULTS: Only a minority of subsets of cells from the tumors of neuroepithelial tissue had the capacity to survive, proliferate, and generate free-floating neurosphere-like BTSs in the simplified serum-free medium. These cells attached to the poly-L-lysine coated coverslips in the serum-supplemented medium and differentiated. The BTSCs were CD133 and nestin positive. The rate of CD133 positive cells in the tumor specimens was (21 +/- 6.2)% - (38 +/- 7.0)%. CONCLUSION: A new simplified culture system for the isolation of BTSCs is established. The tumors of human neuroepithelial tissue contain CD133 and nestin positive tumor stem cells which can be isolated, proliferate and differentiate in vitro and give rise to brain tumor spheres. This tumorigenic subset may provide both a platform for brain tumor research and a target for clinical treatment.

[Thrombin preconditioning reduces brain injury caused by intracerebral infusion of high dose thrombin].

OBJECTIVE: To explore the effect of the cerebral thrombin preconditioning on the thrombin-induced brain edema, to detect the expression of tumor necrosis factor-alpha (TNF-alpha), and to analyse the relationship between TNF-alpha and the thrombin-induced brain edema. METHODS: Forty SD rats were randomly divided into a ST group and a TT group. The rats received 50 L saline (ST group) or 1 U thrombin infusion (TT group), and received the second infusion (10 U thrombin) 24 h later. The rats were sacrificed at 24 and 72 h after the second infusion in order to examine the changes of brain water and sodium contents as well as the expression of TNF-alpha in the brain. RESULTS: The brain water and sodium contents in the ST group were significantly higher than those on the TT group, and those on the 1st day were higher than those on the 3 th day. The positive expression of TNF-alpha and in the change of water content were identical in the TT group and the ST group. CONCLUSION: Thrombin preconditioning can alleviate the thrombin-induced brain edema. The increase of TNF-alpha expression after thrombin treatment may be related to the thrombin-induced brain edema.

[Mononostril-septum-transsphenoidal approach for pituitary adenoma].

OBJECTIVE: To summarize the mononostril-septum-transsphenoidal approach for pituitary adenoma. METHODS: The clinical features, operative techniques, and outcome of 36 patients with pituitary adenoma were analyzed retrospectively. RESULTS: Tumors were totally removed in 28 cases, and subtotally resected in 8 patients. No patient died after the operation. Endocrine symptom of 31 patients returned to the normal level, the symptom of the other 5 cases were improved. Thirty patients with visual field defects recovered after the operation. Cerebrospinal fluid leakage occurred in one patient, and was cured with conservative treatment in 2 weeks. CONCLUSION: Mononostril-septum-transsphenoidal approach can make use of the natural space of the nasal cavity, which has many advantages, such as direct approach, short operative time, minimal invasion, and few complications. It is a effective transsphenoidal surgical approach.

[Correlation research between cancer stem cells and the pathological grades of neuroepithelial tumors].

OBJECTIVE: To explore the methods of isolation, culture and identification of brain tumor stem cells (BTSCs) in neuroepithelial tumor tissues in vitro, and to study the correlation between BTSCs and the patholorical grades of neuroepithelial tumors. METHODS: Tumor cells from patients undergoing neuroepithelial tumors excision were acutely dissociated, triturated into single cells, and then seeded into serum-free medium. After the primary brain tumor spheres (BTSs) were generated, they were triturated again and passaged in fresh medium. The expression of Nestin and CD133 of BTSs was detected by immunocytochemistry staining, and the expression of CD133 of tumor specimen sections was detected by immunohistochemistry staining . The expression of CD133 of 46 brain tumors and 5 normal brain tissues were analysed by SABC immunohistochemical staining, and the correlation between the expression and pathological grade of the tumors was analysed. RESULTS: BTSCs from neuroepithelial tumors could be isolated and cultured, and could be generated and passaged in vitro. The expression of Nestin and CD133 could be detected in BTSCs. CD133 could be detected in neuroepithelial tumor tissues, but not in normal brain tissues. There was significant difference between the expression of CD133 and the different grades of tumors (P < 0.01), and there was a positive correlation between the expression of CD133 and the histologic grading of tumors (P < 0.01). CONCLUSION: A small proportion of stem cells have the ability to self-renew in human neuroepithelial tumors, and there is a positive correlation between the expression of CD133 and histologic grading of tumors.

[Isolation and characterization of brain tumor stem cells in human medulloblastoma].

BACKGROUND & OBJECTIVE: Tumor stem cells have been isolated from several kinds of solid tumors, including primary brain tumors such as glioma and medulloblastoma. This investigation was to establish a simplified culture system to isolate and passage brain tumor stem cells (BTSCs) from human medulloblastoma, observe their proliferation and differentiation, and determine the expression of normal neural stem cell antigens, CD133 and Nestin, in BTSCs. METHODS: Eleven specimens of medulloblastoma were acutely dissociated and triturated into single cells without trypsin digestion. The tumor cells were seeded into serum-free DMEM/F12 medium (200,000 viable cells/ml) containing B27 (1:50), basic fibroblast growth factor (bFGF, 20 microg/L), epidermal growth factor (EGF, 20 microg /L), insulin (4 u/L), L-glutamine, and antibiotics. After generation, the primary brain tumor spheres (BTSs) were mechanically dissociated and passaged in the above serum-free medium. The monoclonal formation experiment was performed to determine the proportion of BTSCs in medulloblastoma and to observe the formation of BTSs. The differentiation of BTSCs was induced in mitogen-free DMEM/F12 medium supplemented with 10% fetal bovine serum. The expression of CD133 and Nestin in BTSs was observed with immunofluorescence staining; the distribution of CD133-positive cells in tumor sections was assessed by immunohistochemistry. RESULTS: In each of the 11 specimens, only a minority of medulloblastoma cells showed the capacity of self-renew and proliferation. These BTSCs generated free-floating neurosphere-like BTSs in the simplified serum-free medium. The proportion of BTSCs with monoclonal formation capacity in primary tumor cells was (31.18+/-6.18)%. The BTSCs attached to poly-L-lysine-coated coverslips and differentiated when the serum-supplemented medium was added. The expression of CD133 and Nestin was detected in BTSCs. CD133-positive cells scattered or formed nest-like aggregations in tumor masses, and accounted for (33.06+/-8.57)% of all tumor cells. CONCLUSIONS: BTSCs, with the capacity of self-renew and proliferation and express CD133 and Nestin, are exist in human medulloblastoma. They could be isolated and cultured in the simplified serum-free medium, and their differentiation could be induced in serum-supplemented medium.

[Microsurgical treatment for craniopharyngioma combined transorbital-subfrontal and temporal craniotomy].

OBJECTIVE: To summarize the experience in microsurgical removal of craniopharyngioma using combined transorbital-subfrontal and temporal craniotomy. METHODS: Eighteen patients with craniopharyngioma varied from 3.1 cm to 6.2 cm in diameter. The tumor was located in the suprasellar region in 7 patients, extended to the third ventricle in 6, and down to the intrasellar from the suprasellar region in 4, and in the third ventricle in 1. Complete or partial cystic tumor was seen in 13 patients, and solid tumor in 5, and calcified tumor in 12. All the patients were operated on via combined transorbital subfrontal and temporal approach. The tumor was dissected in the spaces I, II and IV with great attention to the preservation of the perforating arteries from the carotid, posterior communication and anterior choroidal arteries to the structure of the hypothalamus. The solid portion of the tumor was removed by piecemeal. RESULTS: The tumor was totally removed in 14 patients and subtotally in 4. Postoperation, follow-up for 8 to 41 months showed no change in 3 residual tumors and one lost to follow-up. All patients Postoperative Karnofsky scales showed 80 - 90, in 12 patients, 60 - 70 in 5 patients, and 50 in 1. CONCLUSIONS: Combined transorbital-subfrontal and temporal approach can provide an excellent exposure to the sellar region, craniopharyngioma and its surrounding structures. This approach ensures less cerebral retraction for easy access to craniopharyngioma, including other large neoplasm of the middle cranial base with ventricle or posterior cranial base extension. Microsurgical techniques play an important role in removing tumor and preserving hypothalamic function.

[Keyhole microsurgery for acoustic neurinomas through suboccipital retrosigmoid sinus approach].

BACKGROUND AND OBJECTIVE: Acoustic neurinomas is one of the most common benign tumor in central nervous system, the main treatment is surgical resection. This study is to explore keyhole surgery with suboccipital retrosigmoid sinus approach for acoustic tumor resection and discuss how to improve the surgical skill and effect. METHODS: Thirteen cases of acoustic neurinomas from Sept. 2000 to Dec. 2001 in our department were operated using keyhole craniotomy technique through unilateral modified suboccipital retrosigmoid approach with "[symbol: see text]" incision. All patient underwent suboccipital craniotomy in order to maintain anatomical replacement, and the tumors (2.0-4.4 cm in diameter) were removed under microscope. RESULTS: Tumors were completed resected in 11 cases, subtotally removed in 2 cases. Eleven patients had obtained anatomic preservation of the facial nerves. Complete follow-up information was obtained in all patients for a period of 3-15 months after operation. House-Brackmann Score in 8 case were Grade I-II, 4 cases Grade III-IV, one case Grade V. Grades I and II facial nerve function were maintained in 61.5% of cases, measurable hearing was preserved in 53.8% of cases(7 cases), and 38.5% of cases maintained serviceable hearing. No severe permanent operative complications were found and no surgical mortality occurred. CONCLUSIONS: Microsurgery with keyhole craniotomy is a safe and effective method for treatment of patients with small and medium-size acoustic neuromas. The advantages of keyhole suboccipital craniotomy are anatomical replacement, less postoperative complications, and beneficial to patient's mental health.