FRK通过N-cadherin/E-cadherin途径抑制胶质瘤细胞侵袭和迁移

目的探讨抑癌基因FRK(Fyn-related kinase)影响胶质瘤细胞侵袭和迁移的机制。方法将真核表达质粒pcDNA3.0-FRK和对照质粒pcDNA3.0转入人胶质瘤U25l细胞中,western blot技术检测FRK/N-cadherin/E-cadherin蛋白表达,细胞划痕试验检测细胞迁移能力,Transwell侵袭实验检测细胞侵袭能力。结果与对照组比较,转染pcDNA3.0-FRK质粒24 h后U25l细胞侵袭能力下降47%、迁移能力下降64%,差异均有统计学意义(P0.01);转染pcDNA3.0-FRK可以明显增加N-cadherin/E-cadherin的表达。结论FRK可以通过增加N-cadherin/E-cadherin的表达,进而抑制胶质瘤细胞侵袭和迁移能力。     

FRK通过N-cadherin／E-cadherin胶质瘤细胞侵袭和迁移途径抑制

目的探讨抑癌基因FRK（Fyn-relatedkinase）影响胶质瘤细胞侵袭和迁移的机制。方法将真核表达质粒pcDNA3．0-FRK和对照质粒pcDNA3．0转入人胶质瘤U251细胞中,westernblot技术检测FRK／N．cadherin／E—cadherin蛋白表达,细胞划痕试验检测细胞迁移能力,Transwell侵袭实验检测细胞侵袭能力。结果与对照组比较,转染pcl）NA3．0-FRK质粒24h后U251细胞侵袭能力下降47％、迁移能力下降64％,差异均有统计学意义（P〈0．01）;转染pcDNA3．0-FRK可以明显增加N—cadherin／E—cadherin的表达。结论FRK可以通过增加N．cadherin／E—cadherin的表达,进而抑制胶质瘤细胞侵袭和迁移能力。     

FRK通过抑制ERK信号通路对脑胶质瘤细胞增殖作用的研究

目的研究FRK是否通过抑制ERK信号通路进而影响脑胶质瘤细胞的增殖。方法应用PolyJet~(TM)将FRK质粒转染入脑胶质瘤U251细胞中,Western blot(WB)检测转染效率及P-ERK、ERK蛋白水平的变化,EDU实验观察脑胶质瘤细胞增殖能力的变化;用ERK抑制剂PD98059处理U251细胞,WB检测细胞中FRK、P-ERK、ERK的蛋白水平,EDU实验检测脑胶质瘤细胞增殖能力的变化。结果 WB检测显示FRK质粒转染成功,过表达FRK使U251细胞增殖能力降低。过表达FRK降低了P-ERK的蛋白水平,但对ERK总蛋白水平无影响。与对照组相比,ERK抑制剂PD98059组P-ERK的蛋白水平明显降低,但对FRK的蛋白水平无明显影响。ERK抑制剂PD98059处理后,脑胶质瘤U251细胞增殖能力明显降低。结论 FRK可以通过抑制ERK的活性,从而降低脑胶质瘤细胞的增殖。     

胶质瘤干细胞外泌体通过PI3K/Akt信号通路促进胶质瘤U87细胞侵袭、迁移

目的 探讨胶质瘤干细胞（GSCs）外泌体（exo）对胶质瘤U87细胞侵袭、迁移的影响。方法 取对数生长期U87细胞,应用干细胞培养液分离、培养胶质瘤U87细胞中GSCs并提取、鉴定GSCs-exo。取对数生长期U87细胞随机分为4组:对照组和高、中、低GSCs-exo浓度组（分别加入20、40、80 μg/ml的GSCs-exo）。Transwell实验检测U87细胞侵袭能力,划痕实验检测U87细胞迁移能力,免疫印记法检测U87细胞磷脂酰肌醇-3激酶（PI3K）/蛋白激酶B（Akt）、L1CAM的表达水平。结果 成功分离GSCs并获得GSCs-exo。GSCs-exo组U87侵袭细胞数、细胞迁移率明显高于对照组（P<0.05）,L1CAM、PI3K及p-AKt/Akt表达水平明显高于对照组（P<0.05）,而且均呈浓度依赖性（P<0.05）。结论 GSCs-exo可促进胶质瘤侵袭与转移,其机制可能与上调L1CAM、PI3K表达,促进Akt磷酸化有关。     

槐定碱通过Wnt/β-catenin信号通路抑制人胶质瘤U87细胞迁移、侵袭

目的 探讨槐定碱对人胶质瘤U87细胞迁移、侵袭能力的影响及其作用机制。方法 体外培养人胶质瘤U87细胞,加入槐定碱[0 mg/ml（对照组）,1.0 mg/ml,2.0 mg/ml,3.0 mg/ml]共培养24 h,划痕实验检测细胞迁移能力,Transwell小室实验检测细胞侵袭能力,免疫印迹法检测细胞β-catenin、E-cadherin、Vimentin和MMP-9蛋白表达,RT-PCR检测细胞Vimentin、MMP-9 mRNA表达。结果 槐定碱明显抑制U87细胞迁移、侵袭能力（P<0.001）,而且随浓度增加抑制作用明显增强（P<0.001）。槐定碱明显抑制U87细胞β-catenin蛋白、Vimentin和MMP-9 mRNA和蛋白表达（P<0.01）,明显增强E-cadherin蛋白表达（P<0.01）,而且随浓度增加抑制作用明显增强（P<0.01）。结论 槐定碱能抑制人胶质瘤U87细胞的迁移和侵袭,机制可能与抑制Wnt/β-catenin信号通路的活性,阻断细胞上皮间质转化过程。     

胶质瘤中PTEN、FAK与Ki67表达的研究

目的 探讨PTEN、FAK与Ki67在胶质瘤增殖、侵袭过程中的相互作用。方法 应用SP免疫组化法检测了50例不同级别胶质瘤中的三种蛋白表达情况。结果 不同级别的胶质瘤中PTEN、FAK与Ki67的表达水平不同,Ⅱ级与Ⅲ、Ⅳ级肿瘤之间三者表达差异显著;PTEN与FAK、Ki67在胶质瘤中的表达呈负相关。结论提示PTEN表达缺失可引起FAK及Ki67的过表达,从而加强胶质瘤的增殖和侵袭。     

Rac1在脑胶质瘤干细胞迁移和侵袭中的作用

目的 通过特异性的Racl活性抑制剂下调胶质瘤干细胞(GSCs)中Rac1的活性,探讨Rac1在GSCs迁移和侵袭中的作用. 方法 将U251细胞置于无血清DMEM/F12干细胞培养基中培养,免疫磁珠分选法分离GSCs,免疫荧光染色检测CD133鉴定GSCs; Rac1活性实验检测CD133+与CD133-细胞活性,Transwell细胞迁移和侵袭实验评价CD133+与CD133-细胞的迁移和侵袭能力;加入Rac1活性抑制剂NSC23766处理GSCs,活性实验检测细胞Racl活性,Western blotting检测hMena和基质金属蛋白酶9(MMP-9)蛋白的表达;Transwell细胞迁移实验和侵袭实验评价细胞迁移和侵袭能力的改变. 结果 成功培养出悬浮生长的细胞球,可以连续传代并持续表达神经干细胞标志物CD133;CD133+细胞Rac1-三磷酸鸟苷(GTP)表达水平、细胞迁移和侵袭的能力显著高于CD133-细胞,差异有统计学意义(P＜0.05);与对照组比较,NSC23766处理组GSCs的Rac 1-GTP、hMena和MMP-9蛋白表达水平明显降低,迁移和侵袭能力明显减弱,差异有统计学意义(P＜0.05). 结论 GSCs相对于肿瘤中的其他细胞具有更强的迁移和侵袭能力,Rac1对脑GSCs的迁移和侵袭具有调控作用,抑制Rac1活化可能成为治疗恶性胶质瘤的新策略.     

白藜芦醇阻断TGF-β1/Smad信号通路抑制脑胶质瘤细胞迁移侵袭及上皮间质转化

目的 探究白藜芦醇对人脑胶质瘤细胞黏附、迁移、侵袭、上皮间质转化(EMT)及转化生长因子-β 1(TGF-β 1)/Smads信号通路的调控作用。方法 体外培养人脑胶质瘤T98G细胞,分为对照组(不做干预)、实验组(12.5μmol·L^-1白藜芦醇组、25μmol·L^-1白藜芦醇组、50μmol·L^-1白藜芦醇组)、5-氟尿嘧啶组(50 mg·L^-1的5-氟尿嘧啶)、抑制剂组(50μmol·L^-1白藜芦醇+10μmol·L^-1 TGF-β 1/Smad通路抑制剂LY2109761)和激活剂组(50μmol·L^-1白藜芦醇+10μmol·L^-1TGF-β 1/Smad通路激活剂SRI-011381),干预24 h。用细胞计数试剂盒比色(CCK-8)、倒置显微镜、黏附实验、划痕实验、Transwell小室及蛋白印迹(Wb)法对细胞活力、形态、黏附、迁移、侵袭能力及EMT和TGF-β 1/Smad信号通路相关蛋白表达水...     

敲低WTAP表达抑制脑胶质瘤干细胞的增殖、迁移和侵袭

目的 探讨敲低Wilms肿瘤抑制因子（WTAP）表达对脑胶质瘤干细胞增殖、迁移和侵袭能力的影响及机制。方法 从手术切除胶质瘤标本中提取胶质瘤干细胞（GSC）,应用lipofectamine2000转染WTAP shRNA质粒敲低WTAP表达,以shRNA为阴性对照;PCR检测WTAP mRNA水平分析敲低效率;MTT法检测细胞增殖能力,Transwell小室法检测细胞迁移和侵袭能力,免疫印迹法检测细胞AKT磷酸化水平。结果 从胶质瘤标本中成功提取GSC,其SOX2表达显著降低（P<0.001）,而α-SMA和PDGFRb的表达上调（P<0.001）。与阴性对照组相比,WTAP敲低组GSC细胞WTAP mRNA水平明显降低（P<0.05）,细胞增殖、迁移和侵袭能力均显著下降（P<0.05）,细胞AKT的磷酸化水平显著降低（P<0.05）。结论 敲低WTAP表达可抑制GSC的增殖、迁移和侵袭能力,其机制可能与抑制AKT磷酸化有关。     

反义miR-21通过RECK抑制胶质瘤细胞侵袭性生长的体内外研究

目的 探讨胶质瘤细胞LN229中RECK作为miR-21的调控靶点,在胶质瘤侵袭性生长中的作用.方法 将反义miR-21(AS-miR-21)寡核苷酸转染至人脑胶质瘤细胞LN229中.Real-time PCR检测LN229细胞中miR-21的表达量.荧光素酶实验检测miR-21对RECK的调控关系.Transwell实验评价LN229细胞侵袭能力的变化,应用Western blot检测细胞内MMP2/9和RECK蛋白水平的变化,ELISA实验检测培养基中活性MMP2/9的表达量,动物实验评价体内条件下肿瘤侵袭性的变化.结果 Real-time PCR显示转染组中miR-21的表达量与对照组相比下调60%.荧光素酶实验证明RECK是miR-21的靶点.Transwell实验证实胶质瘤细胞侵袭能力下降,Western blot和ELISA实验证实MMP2/9表达降低,动物实验及免疫荧光反映肿瘤侵袭性生长受抑制.结论 反义miR-21通过上调RECK的表达而抑制恶性胶质瘤细胞的侵袭性生长.

Abstract：

Objective To investigate the regulation of miR - 21 on invasion growth of human glioma cells by RECK.Method The human glioma LN229 cells were transfected with AS - miR - 21 or scrambled sequences by Lipofectamine2000.Real time PCR was conducted to detect the expression of miR-21.Luciferase experiment was performed to detect the relationship between miR-21 and RECK.The expression of RECK was evaluated by Western blot.The invasion ability was evaluated by transwell assay and subcutaneous models.Western Blot, ELISA and immunofluorescence were used to estimate the changes of MMP2/9.Results The expression of miR - 21 in LN229 cells decreased after transfection with AS-miR-21. It was proved that RECK was a direct target of miR -21 by luciferase experiment.Meanwhile, the high expression of RECK protein in AS - miR -21 group conformed its important function in this mechanism.Transwell assay demonstrated decreased invasion capability of LN229 cell lines transfected with AS- miR- 21.Western blot, ELISA, and immunofluorescence demonstrated the levels of MMP2/9 were down -regulated in AS -miR -21 group compared with control and scrambled group.Conclusions AS - miR -21 could depress the invasion of glioma cells owing to up - regulating the level of RECK which could inhibit MMP2/9 activities both in vitro and vivo.     

Intersectin1-s is involved in migration and invasion of human glioma cells

Malignant gliomas have a tendency to invade diffusely into surrounding healthy brain tissues, thereby precluding their successful surgical removal. Intersectin1 (ITSN1) as a molecular linker in the central nervous system is well known as an important regulator of endocytosis and exocytosis. ITSN1 has two isoforms: ITSN1-l and ITSN1-s. In this study, we show that siRNA-mediated down regulation of ITSN1-s inhibited migration and invasion of glioma cells. In addition, we demonstrate the possible mechanisms by which ITSN1-s functions in migration and invasion. Several key proteins, including cofilin, LIMK, PAK, FAK, integrin β1, and MMP-9, which are critical for cells migration and invasion, were probably involved in ITSN1-s signaling pathways. These results suggest that ITSN1-s contributes to glioma cells migration and invasion by regulating the formation of cytoskeleton, influencing adhesion and increasing expression of MMP-9. Our results indicate that ITSN1-s is a critical factor in gliomas invasion and identify that ITSN1-s is a new potentially antiinvasion target for therapeutic intervention in gliomas.     

Leptin induces migration and invasion of glioma cells through MMP-13 production

Leptin, the product of the obese gene, plays an important role in the regulation of body weight by coordinating metabolism, feeding behavior, energy balance, and neuroendocrine responses. However, regulation of leptin gene expression in the central nervous system is different from that in the adipocytes. In addition, leptin has been found in many tumor cell lines and has been shown to have mitogenic and angiogenic activity in a number of cell types. Glioma is the most common primary adult brain tumor with poor prognosis because of the spreading of tumor cell to the other regions of brain easily. Here we found that malignant C6 glioma cells expressed more leptin and leptin receptors than nonmalignant astrocytes. Furthermore, it was found that exogenous application of leptin enhanced the migration and invasion of C6 glioma cells. In addition, we found that the expression of matrix metalloproteinase-13 (MMP-13) but not of MMP-2 and MMP-9 was increased in response to leptin stimulation. The leptin-induced increase of cell migration and invasion was antagonized by MMP-13 neutralizing antibody or silencing MMP-13. The up-regulation of MMP-13 induced by leptin was mainly through p38 MAP kinase and NF-kappaB pathway. In addition, migration-prone sublines demonstrate that cells with increasing migration ability had more expression of MMP-13 and leptin. Taken together, these results indicate that leptin enhanced migration and invasion of C6 glioma cells through the increase of MMP-13 production.     

上皮间质转化与胶质瘤侵袭迁移的研究进展

胶质瘤是一种恶性程度高、浸润性生长的颅内原发性肿瘤。肿瘤的侵袭和转移是导致患者预后差的主要原因。上皮细胞-间质细胞转换(EMT)是胶质瘤细胞获得高度迁移和侵入表型特征的关键过程。EMT是一个进化上保守的细胞发育程序,和致癌效应密切相关。该文就EMT与胶质瘤侵袭迁移相关作用机制和靶向治疗进行综述,为胶质瘤的诊疗研究提供理论依据。     

Slit2/Robo1 signaling in glioma migration and invasion

Slit2/Robo1 is a conserved ligand-receptor system, which greatly affects the distribution, migration, axon guidance and branching of neuron cells. Slit2 and its transmembrane receptor Robo1 have different distribution patterns in gliomas. The expression of Slit2 is at very low levels in pilocytic astrocytoma, fibrillary astrocytoma and glioblastoma, while Robo1 is highly expressed in different grades of gliomas at both mRNA and protein levels. Acquisition of insidious invasiveness by malignant glioma cells involves multiple genetic alterations in signaling pathways. Although the specific mechanisms of tumor-suppressive effect of Slit2/Robo1 have not been elucidated, it has been proved that Slit2/Robo1 signaling inhibits glioma cell migration and invasion by inactivation of Cdc42-GTP. With the research development on the molecular mechanisms of Slit2/Robo1 signaling in glioma invasion and migration, Slit2/Robo1 signaling may become a potential target for glioma prevention and treatment.     

Silencing of OTUB1 inhibits migration of human glioma cells in vitro

OTU domain‐containing ubiquitin aldehyde‐binding protein 1 (OTUB1) protein, a deubiquitinating enzyme (DUB) which belongs to the ovarian tumor (OTU) family, was reported to be associated with the development of various malignancies. However, the potential function of OTUB1 in human gliomas was still unclear. In this study, we sought to investigate the function of OTUB1 in the pathological process of gliomas and analyze its related clinical significance. Western blot and immunohistochemistry analyses demonstrated that OTUB1 was overexpressed in glioma tissues, and statistical analysis suggested the expression level of OTUB1 was significantly correlated with the WHO grades of human gliomas (P < 0.05). Moreover, Kaplan–Meier curve also indicated that high expression of OTUB1 was correlated with a poor prognosis. In vitro, silencing OTUB1 retarded the migration ability of glioma cells. Knockdown of OTUB1 increases epithelial‐mesenchymal transition‐related protein E‐cadherin expression, but decreases simultaneously the expression of vimentin and snail. Furthermore, down‐regulated expression of OTUB1 also resulted in decreased expression of some extracellular matrix degradation‐related proteins, such as matrix metallopeptidase (MMP)2 and MMP9. All results suggested that OTUB1 was a valuable marker in the pathogenesis of human gliomas and could be used as a novel biomarker for glioma therapy in the future.     

L1 expressed by glioma cells promotes adhesion but not migration

L1 is an adhesion molecule of the immunoglobulin superfamily expressed by several types of cancer, including gliomas. It has been shown that L1 can act as chemoattractant to glioma cells, while the effects of L1 expressed by glioma cells themselves are unknown to date. We established a C6 rat glioma clone, conditionally expressing murine L1 under control of a tetracycline responsive promoter. In vitro experiments revealed increased adhesion on matrigel as well as increased intercellular adhesion in the presence of L1, whereas no L1-dependent effects on proliferation or migration on either matrigel or myelin were observed. In vivo experiments using transplantation into nude mouse striatum, where L1 expression by glioma cells was regulated by tetracycline via drinking water, did not show effects of L1 on tumor size or brain invasion. Our data suggest that L1 expressed on the surface of glioma cells increases cell-matrix and intercellular adhesion, but has no apparent effects on proliferation and invasion.     

Knockdown of annexin 2 decreases migration of human glioma cells in vitro

Diffuse invasion of brain tissue is a major reason for the poor prognosis of patients with glioblastoma. Annexin 2, a member of the large annexin family of Ca2+ and membrane-binding proteins, is expressed at high protein levels in human gliomas and has been proposed as a marker of glioma malignancy, while its functional role in these tumours is unknown so far. The ability of annexin 2 to interact with the actin cytoskeleton, as well as its potential to bind invasion-associated proteases, suggests that it could participate in invasion-associated processes in human gliomas. Therefore, we analysed here functional consequences of RNA interference-mediated silencing of annexin 2 in U87MG and U373MG human glioma cell lines. While no impact of annexin 2 downregulation on proliferation and adhesion was observed, our analyses revealed that migration of U87MG and U373MG cells was significantly inhibited following annexin 2 depletion. This effect was not related to a compensatory increase of the related annexins 1 or 6. Our findings identify annexin 2 as a potential candidate involved in glioma invasion and support the potential of RNA interference as powerful tool in the decryption of glioma invasion mechanisms.     

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U34...     

Role of high molecular weight extracellular matrix proteins in glioma cell migration

Malignant human gliomas are characterized by an uncontrolled cell proliferation and infiltrative growth within the brain. Complete surgical removal is difficult due to disseminated tumour cells, and the fundamental mechanisms responsible for this spread are poorly understood. An extensive tumour cell movement along blood vessels is frequently observed and this may be due to specific interactions between tumour cell surface receptors and specific extracellular matrix (ECM) components present in conjunction with vascular elements. In order to investigate the influence of ECM on glioma cell migration, three different human glioma cell lines (U-373 MG, A-172 MG and HF-66) were exposed to known ECM components of the basement membrane (laminin, fibronectin and collagen type IV). Cell migration from multicellular spheroids was studied, using a custom-made medium which was prepared by removing the high molecular weight protein fraction (>100 kDa) from newborn calf serum by ultrafiltration. To this medium, the specific ECM components were added. For two of the cell lines (A-172 MG and U-373 MG), laminin was the most potent stimulator of glioma cell migration; the effect of laminin exceeded that evoked by ordinary serum-supplemented medium. For the HF-66 cell line, fibronectin was the most potent stimulator of migration. Western blot analysis showed that the A-172 MG and HF-66 cell lines expressed low amounts of laminin compared with U-373 MG, which showed extensive intrinsic synthesis of this ligand. U-373 MG was the only cell line that migrated in pure filtered medium. The cells stimulated by fibronectin expressed a different morphology from those stimulated by laminin suggesting that specific ECM-receptor binding may activate different cytoskeletal components within the cells. Furthermore, it was shown that there was no difference in the amount of protein synthesis between cells grown in filtered medium and in filtered medium supplemented with different ECM components. This suggests that ECM-induced cell migration is not dependent on a high level of protein synthesis. It is also shown that α3 integrin, which is a receptor-subunit for laminin, fibronectin and collagen type IV, was highly expressed in all cell lines. This study indicates that glioma cells need serum proteins with a molecular weight >100 kDa to migrate in vitro, and that laminin and fibronectin play an important role in this process.