Inhibition of BCNU-Resistance in Glioma Cell Lines by Small Interfering Rna Targeting to Interleukin-8 Gene

It had been found that interleukin-8 （IL-8） was associated with drug resistance. We previously demonstrated that the resistance to 1, 3-bis （2-chloroethyl）-1-nitrosourea （BCNU） of glioma cell line SWOZI was much higher than that of cell line SOWZ2, which were both cloned from the same parental glioma cell line SWO38. In this study, IL-8 was found to be upregulated both in SWOZ1 and SWOZ2-BCNU, a BCNU-resistant glioma cell line. To further investigate the function of IL-8, the BCNU-resistant cell lines SWOZ1 and SWOZ2-BCNU were treated with siRNAs targeting IL-8. The results of quantitative RT-PCR showed that a decreased level of IL-8 mRNA expression in SWOZI and SWOZ2-BCNU for more than 90% compared to negative control and was confirmed by western blot assay （P 〈 0.05） after treated by siRNAs targeting IL-8. Subsequently, the cytotoxicity of BCNU to these cell lines was detected using the cell counting kit-8 assay. As a result, the BCNU resistance was reversed for about 50% both in these two cell lines （P 〈 0.05）. Our data demonstrated that inhibition of IL-8 with specific siRNAs can reverse the BCNU resistant phenotype in glioma cell lines, indicating that IL-8 may play an important role in BCNU-resistance in glioma.     

Alterations in the Cellular Immune Response of Patients with Cerebral Glioma,Benign Intracranial Tumour,and Spontaneous Subarachnoid Haemorrhage Measured In Vitro by the Leucocyte Migration Inhibition Test

Leucocyte migration inhibition in response to ubiquitous antigens was studied in 104 patients as an in vitro indicator of cell-mediated immunity. Patients with cerebral glioma, benign intracranial tumours, and subarachnoid haemorrhage demonstrated impaired inhibition of leucocyte migration compared with control subjects. The greatest impairment occurred in patients with subarachnoid haemorrhage, while the least impairment was seen in patients with glioma. Significant rises in inhibition of leucocyte migration in response to antigen preparations from glioma and normal brain were seen in the early post-operative period in patients with glioma and subarachnoid haemorrhage. Impaired cellular immunity, together with sensitivity of lymphocytes to brain-derived antigens, are features of cerebral disease in general and not specific for glioma.     

血清CXCL9和TIMP-1水平对脑胶质瘤诊断价值的评价

目的 探讨血清中CXC趋化因子家族9(Chemokine C-X-C motif ligand 9,CXCL9)和组织金属蛋白酶抑制剂-1(Tissue inhibitor of metalloproteinases-1,TIMP-1)对脑胶质瘤恶性程度的鉴别价值.方法 使用ELISA法检测低级别和高级别脑胶质瘤组中CXCL9和TIMP-1的含量.结果 与低级别脑胶质瘤组相比较,高级别脑胶质瘤组血清CXCL9和TIMP-1含量均显著升高,且差异具有统计学差异(P＜0.05).CXCL9区分两组的受试者工作特征曲线(Receiver operator characteristic curve,ROC)下面积为0.758(0.723,0.793),对于高级别脑胶质瘤的诊断敏感性和特异性分别为72.7％和67.9％.TIMP-1的ROC曲线下面积为0.722(0.694,0.751),敏感性和特异性分别为70.5％和62.5％.利用二元Logistic回归分析评价血清CXCL9和TIMP-1联合检测的诊断价值,其曲线下面积为0.855(0.792,0.917),敏感性和特异性分别为80.4％和76.1％.CXCL9和TIMP-1的联合诊断与CXCL9和TIMP-1单独检测的诊断价值相比,其曲线下面积有显著提高(P =0.014,P=0.007).结论 CXCL9和TIMP-1的联合诊断是脑胶质瘤患者鉴别诊断的一种潜在的辅助诊断方法.     

PCC0208017, a novel small-molecule inhibitor of MARK3/MARK4, suppresses glioma progression in vitro and in vivo

Gliomas are the most common primary intracranial neoplasms among all brain malignancies, and the microtubule affinity regulating kinases (MARKs) have become potential drug targets for glioma. Here, we report a novel dual small-molecule inhibitor of MARK3 and MARK4, designated as PCC0208017. In vitro, PCC0208017 strongly inhibited kinase activity against MARK3 and MARK4, and strongly reduced proliferation in three glioma cell lines. This compound attenuated glioma cell migration, glioma cell invasion, and angiogenesis. Molecular mechanism studies revealed that PCC0208017 decreased the phosphorylation of Tau, disrupted microtubule dynamics, and induced a G2/M phase cell cycle arrest. In an in vivo glioma model, PCC0208017 showed robust anti-tumor activity, blood–brain barrier permeability, and a good oral pharmacokinetic profile. Molecular docking studies showed that PCC0208017 exhibited high binding affinity to MARK3 and MARK4. Taken together, our study describes for the first time that PCC0208017, a novel MARK3/MARK4 inhibitor, might be a promising lead compound for treatment of glioma.     

TERT启动子突变、ATRX表达水平在人脑胶质瘤病人预后评估中的作用

目的 探讨端粒酶逆转录酶（TERT）启动子突变、α-地中海贫血伴智力低下综合征基因（ATRX）表达水平在人脑胶质瘤病人预后评估中的价值。方法 回顾性分析2016年6月~2018年6月手术治疗的102例人脑胶质瘤的临床资料。术后检测脑胶质瘤组织TERT启动子突变及ATRX表达情况。所有病人术后随访2年。结果 102例中,低级别胶质瘤48例（WHO分级Ⅱ级）,高级别胶质瘤54例（Ⅲ级31例,Ⅳ级23例）。低级别组TERT启动子突变率（37.50%,18/48）明显低于高级别组（59.26%,32/54;P<0.05）,而ATRX表达阳性率（60.42%,29/48）明显高于高级别组（37.04%,20/54;P<0.05）。多因素Cox比例回归风险模型结果显示,TERT启动子突变、ATRX表达阴性是生存预后不良的独立危险因素（P<0.05）。生存曲线分析显示,TERT启动子突变型胶质瘤病人较野生型病人生存期明显缩短（P<0.05）,ATRX表达阴性胶质瘤病人较表达阳性病人生存期明显缩短（P<0.05）。结论 TERT启动子突变、ATRX基因表达对人脑胶质瘤预后评估具有重要价值。     

Hax-1对胶质瘤细胞增殖、迁移和侵袭的作用

目的 探讨造血干细胞特异性相关结合蛋白-1（Hax-1）对胶质瘤细胞增殖、迁移和侵袭的影响。方法 选择2018年9月至2019年6月手术切除并得到术后病理证实的胶质瘤组织35例和颅脑损伤内减压术切除的正常脑组织35例,采用qRT-PCR检测Hax-1 mRNA水平;同时检测胶质瘤细胞系（U87、A172、T98及U343）和人星形胶质细胞（NHAs）Hax-1 mRAN水平。使用Lipofectamine 2000法将Hax-1 siRNAs和阴性对照siRNA转染U87细胞构建Hax-1低表达细胞株,将Hax-1高表达质粒和PCDNA3.1空载质粒转染U343细胞构建Hax-1过表达细胞株,使用蛋白印迹法验证转染效果,采用CCK-8法检测细胞增殖,采用Transwell小室实验检测细胞迁移和侵袭,免疫印迹法检测NF-κB信号通路相关蛋白表达水平（NF-κB、CCND1、C-myc、MMP-2、MMP-9）。结果 胶质瘤组织Hax-1 mRNA水平明显高于正常脑组织（P<0.05）;胶质瘤细胞系（U87、A172、T98及U343）Hax-1 mRNA水平明显高于人星形胶质细胞（P<0.05）,其中U87细胞Hax-1 mRNA水平最高,U343细胞最低。U343细胞转染Hax-1过表达质粒后,Hax-1蛋白水平显著增高（P<0.05）,细胞增殖、侵袭、迁移能力均明显增强（P<0.05）,NF-κB p65及IκBα蛋白磷酸化水平以及CCND1、C-Myc、MMP-2、MMP-9蛋白表达水平明显增高（P<0.05）。U87细胞转染Hax-1 siRNA后Hax-1蛋白水平显著降低（P<0.05）,细胞增殖、侵袭、迁移能力均明显降低（P<0.05）,NF-κB p65及IκBα蛋白磷酸化水平以及CCND1、C-Myc、MMP-2、MMP-9蛋白表达水平明显降低（P<0.05）。结论 胶质瘤组织Hax-1呈高表达,明显促进肿瘤细胞增殖、侵袭和迁移,其机制可能与激活NF-κB信号通路有关。     

胶质瘤组织UCH37表达水平与病人预后的相关性

目的 探讨胶质瘤组织泛素化羧基末端水解酶37（UCH37）表达水平与病人预后的相关性。方法 选取2017年6月至2019年6月手术切除的脑胶质瘤组织104例和瘤旁脑组织64例,采用免疫组化法检测组织UCH37表达水平,根据免疫组化染色评分分成高表达组、低表达组;采用PCR法检测组织UCH37 mRNA水平。术后随访18个月,记录死亡、生存情况。结果 104例中,死亡20例,存活84例;UCH37高表达62例,低表达42例。脑胶质瘤组织UCH37高表达率（59.62%）明显高于瘤旁组织（17.19%;P<0.05）,而且脑胶质瘤组织UCH37 mRNA表达量明显高于瘤旁组织（P<0.05）。多因素logistic回归分析显示UCH37高表达是脑胶质瘤术后死亡的独立危险因素（P<0.05）。UCH37高表达组中位生存期（9个月）较低表达组（13个月）明显缩短（P<0.05）。ROC曲线分析显示,UCH37 mRNA评估病人术后死亡的最佳界值为2.725,曲线下面积为0.797（95%置信区间0.695~0.899）,敏感度为70.00%,特异度为70.10%。结论 脑胶质瘤UCH37呈高表达,表达水平越高,预后越差。肿瘤组织UCH37 mRNA水平检测对病人预后有一定评估价值。     

胚胎干细胞关键因子Nanog在胶质瘤中的表达及意义

目的 探讨胚胎干细胞关键因子Nanog在人胶质瘤中的表达及意义。方法 通过免疫荧光双标技术,分析40例胶质瘤组织内胚胎干细胞关键因子Nanog与肿瘤干细胞相关基因Nestin、CD133及胚胎干细胞全能性相关基因Oct4 的共表达情况,并通过RT-PCR、Western blotting技术分析胶质瘤组织内Nanog与脑胶质瘤恶性程度之间的关系。结果 Nanog在胶质瘤组织中的表达随着胶质瘤病理级别增高而升高,而且超过50%的Nanog阳性细胞同时表达Nestin和CD133。95%以上的Nanog阳性细胞都同时表达胚胎干细胞全能性相关基因Oct4。结论 胶质瘤组织中Nanog多表达在肿瘤干细胞中,与胶质瘤的恶性程度呈正相关,在胶质瘤的发生、发展过程中起着重要作用,为研究胶质瘤的起源及胶质瘤的诊断和预后判断提供帮助。     

miR-6858 plays a key role in the process of melatonin inhibition of the malignant biological behavior of glioma

MicroRNAs (miRNAs), small non-coding RNA molecules with a length of 18–25 nucleotides, have been shown to be involved in mediating various malignant properties of GBM, including growth, invasion and angiogenesis. Here, we investigated whether miRNAs might be involved in mediating the suppression of malignant properties of GBM by melatonin (MEL), an amine hormone secreted by the pineal gland. Sequencing was performed to screen specifically for miRNAs induced by MEL in U87 and an orthotopically xenografted primary GBM cell line, GBM#P3. MiR-6858-5p was the most significantly up-regulated miR in GBM cell lines in response to MEL (~5 × ). Transfection of a mimic of miR-6858-5p into both cell lines led to a decrease in viability of ~ 50% at 72 h, confirming a suppressive role for miR-6858-5p in GBM. In contrast, an inhibitor of miR-6858-5p rescued GBM cells from MEL suppression of proliferation, migration and invasion. Analysis using Targetscan yielded candidate mRNAs targeted by miR-6858-5p, some of which are involved in the SIRT/AKT signaling pathway. In cells transfected with a mimic or an inhibitor of miR-6858-5p, levels of SIRT3 and downstream components of the AKT signaling pathway were suppressed or up-regulated, respectively, both in vitro and in an in vivo orthotopic xenograft model. Our results elucidated a novel molecular mechanism underlying MEL suppression of GBM, highlighting a role for miRNAs, and provide a potential therapeutic strategy for GBM.