MiR-128-3p靶向抑制HOXA5调控多形性胶质母细胞瘤的增殖、侵袭与凋亡

目的 探索HOXA5在胶质母细胞瘤（GBM）中高表达的原因及miR-128-3p调控胶质母细胞瘤进展的分子机制。方法 通过慢病毒转染上调或下调U87细胞中miR-128-3p的表达水平,再利用蛋白质免疫印迹法检测HOXA5的表达水平的变化来探索miR-128-3p与HOXA5在GBM中表达的相关性。利用双荧光素报告基因实验验证miR-128-3p对HOXA5的靶向抑制关系。利用miR-128-3p与HOXA5过表达的质粒转染U87细胞进行拯救实验,通过CCK-8、Transwell、流式细胞学分析与裸鼠体内实验验证miR-128-3p调控GBM增殖、侵袭及凋亡方面的分子机制。结果 上调U87细胞中miR-128-3p表达后HOXA5的表达水平显著下降,下调U87细胞中miR-128-3p表达后HOXA5表达水平明显升高（P<0.05）,两者表达呈显著负相关。miR-128-3p可靶向结合HOXA5基因的3’UTR区并抑制HOXA5表达。miR-128-3p+Control组U87细胞增殖、侵袭及抗凋亡能力显著下降。结论 miR-128-3p可通过靶向抑制HOXA5负向调控GBM细胞的增殖、侵袭及抗凋亡能力,HOXA5在GBM中呈高表达与miR-128-3p表达水平降低有关。     

KIF20A对胶质瘤细胞增殖、迁移和侵袭、凋亡及细胞周期分布的影响

目的:研究KIF20A对胶质瘤U87细胞系增殖、侵袭、迁移、凋亡及细胞周期分布的影响。方法:利用 RNA干扰技术下调胶质瘤细胞系 U87中 KIF20A 的表达。RT-qPCR 和 Western blot 分别检测 KIF20A 在mRNA 和蛋白水平上的表达。CCK-8 实验检测 U87细胞增殖能力的变化，Transwell 实验检测U87细胞迁移及侵袭能力的变化，流式细胞术检测 U87细胞凋亡及细胞周期的变化。结果:RT-qPCR和Western blot 结果显示 siRNA-KIF20A显著下调 KIF20A 的表达，CCK-8和Transwell 实验显示下调 KIF20A 的表达显著抑制了U87细胞的增殖、迁移及侵袭能力，流式细胞术结果显示下调 KIF20A 的表达显著促进了U87细胞的凋亡，诱导细胞周期阻滞在G0/G1期。结论:下调KIF20A的表达抑制胶质母细胞瘤细胞的增殖、迁移及侵袭，促进胶质母细胞瘤细胞的凋亡并诱导细胞周期G0/G1期阻滞。     

新型 CDK7抑制剂对人胶质瘤细胞生物学功能的影响

目的：探究新型 CDK7抑制剂 THZ1对人胶质瘤细胞系 U87及 U251增殖、凋亡、侵袭能力的影响及细胞周期的阻滞作用。方法：将 U87及 U251细胞分成对照组及实验组,实验组加入不同浓度的 THZ1处理后,以 CCK －8法及平板克隆形成试验分析细胞增殖;Transwell 侵袭试验观测各组侵袭能力的变化;流式细胞仪检测各组细胞周期分布及凋亡情况;Western blot 实验检测凋亡相关蛋白 Caspase －3的表达量。结果：THZ1抑制 U87及 U251的增殖,根据实验结果用 SPSS 20．0分析出72小时药物 IC50,U87细胞为83．1nmol／L、U251细胞为13．7nmol／L。平板克隆形成试验结果显示低浓度药物即可明显抑制克隆形成。THZ1降低U87细胞的侵袭性。THZ1促进 U251细胞凋亡,Caspase －3表达增加。THZ1阻滞 U87及 U251细胞周期进展,G2期细胞显著增加。结论：THZ1能够抑制胶质瘤细胞 U87及 U251的增殖,促进凋亡,抑制侵袭,并阻滞细胞周期进展。     

ADAM17对U87MG胶质瘤细胞增殖与侵袭的影响及作用机制

目的 探讨ADAM17在U87MG细胞增殖与侵袭中的作用及机制.方法 收集人脑胶质瘤组织65例(肿瘤组)与正常脑组织13例(对照组),采用免疫组化、RT-PCR法,检测ADAM17蛋白及mRNA的表达;利用siRNA干扰敲减或ADAM17激活剂(PMA)过表达ADAM17,并予以PI3K抑制剂抑制相关信号通路,MTT及Transwell实验检测各组U87MG细胞的增殖与侵袭变化,Western blot检测ADAM17、p-AKT及AKT蛋白变化.结果 胶质瘤组织中ADAM17mRNA及蛋白水平均高于正常脑组织;与对照组相比,siRNA干扰后,低表达组U87MG细胞的增殖与侵袭能力下降,而激活剂PMA增强ADAM17后,过表达组细胞增殖与侵袭能力增加,差异有统计学意义(P＜0.05);敲减及过表达AD-AM17可导致PI3 K/AKT信号通路下游蛋白发生变化.结果 ADAM17可通过激活PI3K/AKT信号通路促进U87MG细胞增殖与侵袭,有望为胶质瘤的治疗找到新突破点.     

PI3K／AKT信号通路调控MKP3表达促进胶质母细胞瘤细胞增殖的实验研究#br# #br#

目的探讨MKP3在胶质母细胞瘤（GBM）中表达以及MKP3高表达对GBM细胞增殖的影响及其分子作用机制。 方法利用TCGA数据库分析GBM中MKP3表达及不同MKP3表达患者的生存情况;选取GBM细胞U 87 MG分别转染si MKP3和si NC,利用MTT实验和EdU法流式细胞术检测U 87 MG细胞增殖的变化情况;Western blotting检测p AKT、AKT、MKP3及GAPDH的蛋白表达; 1 μmol／L PI3K特异性抑制剂以及pCDH MKP3过表达质粒验证PI3K／AKT MKP3轴在U87 MG细胞增殖中的作用。 结果TCGA数据库分析显示,GBM组织中的MKP3表达显著高于正常组织,差异有统计学意义（P＜005）;MKP3高表达患者生存率显著低于MKP3低表达患者,差异亦有统计学意义（P＜005）。 MTT法结果显示,si MKP3组细胞增殖活力为0431±0116,显著低于Si NC组的0986±0056（P＜005）。EdU法结果显示,沉默MKP3表达可显著抑制U 87 MG细胞的增殖。PI3K／AKT信号通路抑制能够明显阻滞U 87 MG细胞的增殖,过表达MKP3后可使 U 87 MG细胞增殖能力基本恢复。过表达MKP3并不会引起AKT的磷酸化增加。 结论PI3K／AKT信号通路通过上调MKP3表达可以诱导U87 MG细胞增殖,促进GBM发生发展。     

miR-153靶向ZEB2抑制胶质母细胞瘤侵袭与转移

目的:探究miR-153对人胶质母细胞瘤（glioblastoma,GBM）侵袭转移的影响及相关作用机制。方法:利用qRT-PCR检测miR-153在GBM中的表达;将miR-153转染至胶质瘤U251细胞后,应用qRT-PCR 验证转染效率;应用Western blot、Transwell及划痕实验检测U251细胞的上皮间质转化、细胞侵袭及转移能力的变化;应用qRT-PCR及Western blot检测ZEB2的mRNA和蛋白表达;利用质粒转染技术过表达miR-153后,同时过表达ZEB2,再应用Transwell及划痕实验检测U251细胞的侵袭及转移能力的变化。结果:与对照相比,miR-153在GBM中低表达;过表达miR-153显著抑制胶质瘤U251细胞的上皮间质转化、细胞侵袭及转移,并能够抑制U251细胞中ZEB2的蛋白表达;ZEB2过表达有效阻断了miR-153抑制U251细胞侵袭及转移的作用。结论:miR-153能够靶向下调ZEB2,进而抑制胶质瘤U251细胞上皮间质转化及细胞侵袭转移。     

STIP1在脑胶质瘤组织中的表达及对胶质瘤细胞株增殖、凋亡、侵袭的影响

目的:检测磷酸化应激诱导蛋白1（stress-induced phosphoprotein 1，STIP1）在胶质瘤组织和正常脑组织中的表达差异，探讨STIP1对胶质瘤细胞株U87、U251细胞增殖、凋亡及侵袭能力的影响。方法:运用实时定量聚合酶链式反应(qRT-PCR)、免疫组化及蛋白印迹检测胶质瘤组织和正常脑组织中STIP1的表达；小干扰RNA（STIP1-siRNA）转染U87、U251细胞株后，使用噻唑蓝(MTT)法、流式细胞仪和Transwell小室观察细胞的增殖、凋亡和侵袭能力。结果:STIP1在胶质瘤Ⅳ级组织中的表达明显高于正常脑组织，但在胶质瘤Ⅱ、Ⅲ级中的表达与正常脑组织无明显差异。U87、U251细胞株转染STIP1-siRNA后，细胞增殖和侵袭能力明显受到抑制，而细胞凋亡率明显上升。结论:STIP1在胶质母细胞瘤组织中高表达，下调STIP1可以抑制胶质瘤细胞株U87和U251的增殖，阻滞细胞周期，促进其细胞凋亡，并可抑制其侵袭力。     

靶向Survivin基因的短发卡RNA在体内对U251细胞移植瘤的影响

Surivin基因属于凋死蛋白抑制因子（inhibitor of apoptosis,IAP）家族，具有抗细胞凋亡、促细胞增殖和促血管形成等多种生物学活性，其在脑胶质瘤发生和发展过程中的作用尚不完全明确，本研究结盟在观察稳定转染靶向Survivin基因的特异性短发卡RNA（shRNA）真核表达载体对人脑胶质母细胞瘤U251细胞裸鼠体内肿瘤生长和血管形成的影响。方法：将U251细胞，稳定转染Survivin基因shRNA真核表达载体pWHI-SR的U251-SR细胞，以及稳定转染空载体pWHI的U251-P细胞，分别接种于裸鼠背部皮下，建立人脑胶质瘤裸鼠皮下移植瘤模型，定期观察肿瘤生长情况，测定肿瘤体积、绘制肿瘤生长曲线，观察45天后处死动物、称瘤重，采用免疫组化SABC法检测Survivin、增殖细胞核抗原（proliferating cell muclear antigen，PCNA）以及八因子相关抗原（factor Ⅷ related antigen,FⅧRAg）在各组肿瘤标本中的表达，采用TUNEL法检测凋亡细胞，分别计算各组肿瘤标本的增殖指数（proliferative index,PI），凋亡指数（apoptotie index,AI）以及微血管密度（microvessel density,MVD）。结果：与U251、U251-SR组肿瘤标本，U251-SR组裸鼠肿瘤形成时间延迟，肿瘤生长缓慢，肿瘤体积及瘤重均明显减小（P〈0.01）；U251-P组裸鼠相丝，U251-SR组肿瘤标本Survivin蛋白表达明显下调：PI和MVD明显减少，AI明显升高（P〈0.001）。结论：靶向Survivin基因的sbRNA能够在体内明显抑制U251细胞的肿瘤生长和血管形成，Survivn基因可作为脑胶质瘤基因治疗的一个良好靶点，而RNA干涉（RNA interference,RNAi）技术可为脑胶瘤基因治疗的一个良好靶点，而RNA干涉（RNA interference，RNAi）技术可为脑胶质瘤的基因治疗提供新的重点手段。     

ZDHHC12在胶质母细胞瘤中通过YAP1调控肿瘤特性

背景和目的：胶质母细胞瘤（glioblastoma,GBM）是目前常见的恶性脑部肿瘤之一,但GBM相关发病机制仍不完全清楚。本研究旨在分析锌指DHHC结构域蛋白（zinc finger DHHC domain-containing protein,ZDHHC）12对Yes相关蛋白1（Yes-associated protein 1,YAP1）的调控,以及ZDHHC12/YAP1轴对GBM肿瘤特性的调控。方法：在癌症基因组图谱（The Cancer Genome Atlas,TCGA）数据库以及基因型-组织表达（Genotype-Tissue Expression,GTEx）数据库中分析ZDHHC12分别在正常脑组织及GBM中的表达情况,运用蛋白质印迹法（Western blot）以及实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）检测,分析在U87、U251以及人正常星形胶质细胞（NHA）的mRNA表达及蛋白水平。在U87和U251两种GBM细胞系中通过设计的小干扰RNA（small interfering RNA,siRNA）敲低ZDHHC12,并验证YAP1蛋白水平的变化。通过免疫共沉淀（co-immunoprecipitation,Co-IP）验证内源性及外源性ZDHHC12及YAP1的相互关系。通过细胞计数试剂盒-8（cell counting kit-8,CCK-8）实验、细胞平板克隆形成实验、划痕实验分析GBM细胞敲低ZDHHC12后在增殖及迁移能力上发生的改变。检测在敲低ZDHHC12后GBM细胞系中上皮-间质转化（epithelial-mesenchymal transition,EMT）标志物变化。分析ZDHHC12对GBM患者预后的影响。并检测在不同组织样本中ZDHHC12和YAP1蛋白水平。结果：TCGA数据库及GTEx数据库分析结果显示,ZDHHC12在GBM中的表达量明显高于正常脑组织（P<0.01）,GBM细胞系中ZDHHC12的mRNA表达及蛋白水平高于NHA细胞系。U87和U251两组GBM细胞系中ZDHHC12的敲低会引起YAP1蛋白水平的降低。Co-IP实验验证了ZDHHC12及YAP1蛋白的相互关系。敲低ZDHHC12的表达能够显著抑制GBM细胞的增殖和迁移能力,差异有统计学意义（P<0.05）。ZDHHC12的敲低也可以引起EMT相关标志物的改变。YAP1的回复可以逆转敲低ZDHHC12所引起的GBM肿瘤特性变化。TCGA数据库中,ZDHHC12的表达也与GBM患者的预后密切相关。在组织中ZDHHC12的表达也与YAP1表达呈高度正相关。结论：ZDHHC12在GBM中高表达且与YAP1呈正相关,ZDHHC12/YAP1轴可以调控GBM相关肿瘤特性。     

SRC抑制胶质母细胞瘤的增殖、迁移、侵袭和干性维持的分子机制

目的 研究SRC在胶质母细胞瘤发生发展中的作用,并初步探讨可能的分子机制。方法  采用生物信息学的方法分析SRC在胶质母细胞瘤中的表达变化;利用shRNA下调胶质瘤母细胞系U87MG中SRC的表达,通过RT-PCR和免疫印迹法验证其抑制效率,并筛选出稳定干涉的细胞株;采用WST-1法、划痕愈合实验和Transwell迁移实验检测SRC shRNA干涉后细胞增殖、迁移和侵袭能力的变化;利用干细胞培养液筛选出SRC shRNA稳定干涉的胶质瘤干细胞,观察SRC shRNA对肿瘤干细胞干性的影响;利用细胞免疫荧光法观察干性基因SOX2的表达变化。结果 在胶质母细胞瘤标本中SRC的表达水平高于对照组,筛选到两条有效的SRC shRNA序列;通过shRNA下调SRC的表达后可以显著抑制胶质瘤母细胞U87MG的增殖、迁移、侵袭和肿瘤干细胞干性维持,并且可以明显抑制SOX2的表达。结论 SRC通过调控胶质母细胞瘤的增殖、迁移、侵袭和干性维持影响其发生发展,其对干性维持的作用可能是通过影响SOX2的表达实现的。     

Involvement of EGFR,ERK-1,2 and AKT-1,2 Activity on Human Glioma Cell Growth

GBM (Glioblastoma multiforme) is the  most prevalent and lethal primary brain tumor. Gene therapy is one of the promising approaches and  involves the delivery of genetic therapeutic molecules for specific antitumour response/activity. miRNAs can regulate the cell biology functions including replication, cell growth, and apoptosis by regulating gene expression. In this study, we found that down-regulation of miR-4731 expression occurred in GBM cells. We further determined that miR-4731 behaved as a tumor suppressor by inhibiting GBM cell proliferation. We further investigated the molecular mechanisms of miR-4731 and EGFR, ERK-1,2 and AKT-1,2 in GBM cell lines U87 and U251. The in vitro ectopic expression of miR-4731 affected cell proliferation, migration, and invasion of U87 and U251 cells. Luciferase reporter assays validated that miR-4731 targeted the 3′-untranslated region (3′-UTR) of EGFR. In conclusions, we identified that miR-4731 plays a tumor suppressor role in GBM cell proliferation and migration by targeting EGFR expression, and miR-4731 may act as a novel biomarker for early diagnosis or therapeutic target of GBM.     

Role of Wnt5a in the proliferation of human glioblastoma cells

Wnt5a operates as either a tumor suppressor or a tumor stimulator, according to tumor type. The functions of Wnt5a in human glioblastoma (GBM) have yet to be determined. We initially evaluated the expression of Wnt5a in human glioma. The results of immunohistochemical analyses have revealed that Wnt5a expression was higher in human GBM than in normal brain tissue and low-grade astrocytoma. In order to assess the role of Wnt5a on proliferation in human glioblastoma cells, we employed U87MG and GBM-05, a newly established GBM cell line. GBM-05 was established from a patient diagnosed with GBM. GBM-05 cells were shown to express Nestin, but did not express GFAP and Map2ab. GBM-05 cells formed infiltrating brain tumors after being intracerebrally transplanted into nude mice, and xenotransplanted GBM-05 cells were observed to differentiate into neuronal and astrocyte lineages. Wnt5a expression in the xenotransplanted tumors was higher than that detected in the surrounding brain tissues. The overexpression of Wnt5a increased the proliferation of GBM-05 and U87MG in vitro. By way of contrast, the downregulation of Wnt5a expression as the result of RNA interference reduced proliferation from GBM-05 and U87MG cells in vitro, and reduced tumorigenicity in vivo. Our data indicate that Wnt5a signaling is an important regulator in the proliferation of human glioma cells.     

Expression and role of PTV1 lncRNA in glioma cells progression

Objective The aim of this study was to investigate the expression of PTV1 lncRNA in gliomas and the mechanism of its interaction with miR-203a.Methods U87 and U251 cells were cultured stably and transfected with sh-PTV1 or ov-PTV1, respectively. The proliferative activity of U87 and U251 cells was detected and the transplanted tumor model nude mice were divided into U87 and U251 groups. U87-sh and u251-ov cells were injected into the armpit, then miR-203a mic and miR-203a inhibitors were administered to detect the changes in the expression of tumor-related proteins. Results The relative expression of PTV1 in gliomas was significantly higher than that in normal brain tissues, while in GBM it was significantly higher than that in low-grade gliomas. Knockdown of PTV1 significantly inhibited the proliferation of U87 cells, resulting in fewer cell clones; overexpression of rPTV1 significantly promoted the proliferation of U251 cells, resulting in more cell colonies. The dual Luciferase Reporter assay showed that SP2 was a potential target of miR-203a. When U87 cells were treated with a miR-203a mimic, the expression of SP2 decreased; and when U251 cells were treated with a miR-203a inhibitor, the expression of SP2 increased significantly. SP2 was overexpressed in u87-sh cells and the proliferation, migration, and invasion of u87-sh cells were significantly enhanced. U251-ov cells showed the opposite trend. Compared with the control group mice, the tumor volume in u87-sh group mice was significantly smaller and the positive rate of SP2 in tumor tissue was significantly lower. After administration of the miR-203a inhibitor, the tumor volume increased gradually and the positive rate of SP2 increased significantly, while u251-ov mice showed the opposite trend. Conclusion lncRNA PTV1 can be used as a molecule to interfere with miR-203a expression in order to downregulate SP2 and to promote the proliferation and invasion of glioma cells. lncRNA PTV1 may be a new biomarker and therapeutic target for glioma.     

Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression

Although rodent glioblastoma (GBM) models have been used for over 30 years, the extent to which they recapitulate the characteristics encountered in human GBMs remains controversial. We studied the histopathological features of dog GBM and human xenograft GBM models in immune-deficient mice (U251 and U87 GBM in nude Balb/c), and syngeneic GBMs in immune-competent rodents (GL26 cells in C57BL/6 mice, CNS-1 cells in Lewis rats). All GBMs studied exhibited neovascularization, pleomorphism, vimentin immunoreactivity, and infiltration of T-cells and macrophages. All the tumors showed necrosis and hemorrhages, except the U87 human xenograft, in which the most salient feature was its profuse neovascularization. The tumors differed in the expression of astrocytic intermediate filaments: human and dog GBMs, as well as U251 xenografts expressed glial fibrillary acidic protein (GFAP) and vimentin, while the U87 xenograft and the syngeneic rodent GBMs were GFAP⁻ and vimentin⁺. Also, only dog GBMs exhibited endothelial proliferation, a key feature that was absent in the murine models. In all spontaneous and implanted GBMs we found histopathological features compatible with tumor invasion into the non-neoplastic brain parenchyma. Our data indicate that murine models of GBM appear to recapitulate several of the human GBM histopathological features and, considering their reproducibility and availability, they constitute a valuable in vivo system for preclinical studies. Importantly, our results indicate that dog GBM emerges as an attractive animal model for testing novel therapies in a spontaneous tumor in the context of a larger brain.