miR-125b对胶质瘤细胞侵袭能力影响的体内研究

目的探讨体内miR-125b表达对人脑胶质瘤细胞侵袭力的影响及其可能的机制。方法培养原代胶质瘤细胞,分别转染miR-125b mimics和miR-125b inhibitor慢病毒载体,上调或下调细胞中miR-125b表达水平,种植乳鼠皮下,Transwell试验评价胶质瘤细胞侵袭能力的变化,Western Blot验证侵袭相关基质金属蛋白酶(MMP)-2、MMP-9及RECK和TIMP3蛋白的表达。结果转染miR-125b mimics能有效提高原代胶质瘤细胞中miR-125b的表达,miR-125b inhibitor有效降低miR-125b的表达;体内实验表明miR-125b mimics及inhibitor对胶质瘤侵袭能力无明显影响,不影响侵袭相关MMP-2、MMP-9及RECK、TIMP3蛋白的表达。结论体内miR-125b表达水平与原代胶质瘤细胞的侵袭能力无明显相关。     

MiR-29a-Mediated CD133 Expression Contributes to Cisplatin Resistance in CD133<Superscript>+</Superscript> Glioblastoma Stem Cells

CD133 positive (CD133⁺) cells are cancer stem cells in glioblastoma that are associated with poor prognosis and resistance to radiotherapy. However, the role of CD133 in chemoresistance is inconclusive, although recent studies suggest that increased CD133 expression may lead to increased cisplatin resistance under certain circumstances. In this study, we further explored the mechanism underlying CD133-mediated cisplatin resistance in glioblastoma stem cells. We sorted human glioblastoma T98G and U87MG cells into CD133⁺ and CD133^? pools and measured apoptosis and CD133 expression levels in response to cisplatin treatment. We predicted candidate microRNAs that might target CD133 and assessed their levels in cisplatin-treated CD133⁺ cells. Finally, we overexpressed miR-29a in CD133⁺ cells and tested its effects in cisplatin-mediated apoptosis and survival of CD133⁺ tumor bearing mice receiving cisplatin treatment. We found that CD133⁺ glioblastoma stem cells showed more resistance to cisplatin treatment. Cisplatin increased CD133 expression by suppressing miR-29a levels. MiR-29a overexpression improved sensitivity of cisplatin in CD133⁺ cells and significantly suppressed tumor growth in CD133⁺ tumor bearing mice in response to cisplatin treatment. Our data show that miR-29a ameliorates CD133-mediated chemoresistance in glioblastoma stem cells, suggesting it as a potential therapeutic target for treating glioblastoma.     

miR-145 Inhibits Migration and Invasion of Glioma Stem Cells by Targeting ABCG2

Despite advances in clinical therapies and technologies, the prognosis for patients with malignant glioma is poor. Our previous research demonstrated that glioma stem cells (GSCs) were crucial for glioma malignancy and accelerated tumor migration and invasion. The migration and invasion of malignant glioma cells into the surrounding normal brain tissues cause the poor outcome. miR-145, a miRNA found to be expressed in neurons, was recently found to have reduced expression in glioblastoma multiforme tumors. And miR-145 loss in glioma cells led to increased cell proliferation and invasion. However, its function on the migration and invasion of GSCs was still unknown. In this study, we aimed to identify the effects and mechanisms of miR-145 on the migration and invasion of GSCs. Our investigations revealed that miR-145 was low expressed in malignant glioma tissues and their corresponding GSCs. Knockdown of miR-145 in vitro could enhance the migration and invasion of GSCs, while up-regulation of miR-145 had the opposite effects. Further investigation of the potential mechanism demonstrated that the function of miR-145 in regulating the migration and invasion of GSCs is mediated by its targeting of ABCG2 mRNA. ABCG2 is an ATP-binding cassette transporter protein, which was identified to be overexpressed in GSCs and higher-grade glioma tissues. We found that miR-145 was negative correlated with ABCG2 levels in GSCs, and reduction in ABCG2 expression decreased the cell migration and invasion of GSCs. Further, a luciferase reporter proved that ABCG2 was a direct target of miR-145 in GSCs. Thus, these findings underscore the potential of miR-145 to regulate the migration and invasion of GSCs through targeting ABCG2.     

MicroRNA miR-133b is essential for functional recovery after spinal cord injury in adult zebrafish

MicroRNAs (miRNAs) play important roles during development and also in adult organisms by regulating the expression of multiple target genes. Here, we studied the function of miR-133b during zebrafish spinal cord regeneration and show upregulation of miR-133b expression in regenerating neurons of the brainstem after transection of the spinal cord. miR-133b has been shown to promote tissue regeneration in other tissue, but its ability to do so in the nervous system has yet to be tested. Inhibition of miR-133b expression by antisense morpholino (MO) application resulted in impaired locomotor recovery and reduced regeneration of axons from neurons in the nucleus of the medial longitudinal fascicle, superior reticular formation and intermediate reticular formation. miR-133b targets the small GTPase RhoA, which is an inhibitor of axonal growth, as well as other neurite outgrowth-related molecules. Our results indicate that miR-133b is an important determinant in spinal cord regeneration of adult zebrafish through reduction in RhoA protein levels by direct interaction with its mRNA. While RhoA has been studied as a therapeutic target in spinal cord injury, this is the first demonstration of endogenous regulation of RhoA by a microRNA that is required for spinal cord regeneration in zebrafish. The ability of miR-133b to suppress molecules that inhibit axon regrowth may underlie the capacity for adult zebrafish to recover locomotor function after spinal cord injury.     

恶性胶质瘤细胞在不同培养液和不同时期中CD133阳性细胞比率的变化

目的:观察不同培养液及不同培养时期等因素对体外培养人脑胶质瘤细胞CD133阳性(CD133+)细胞比率的影响.方法:用干细胞培养液(含EGF、FGF-2、B27)培养6例原代胶质瘤细胞和3例对照细胞株,用流式细胞仪检测培养第0、3、7,28、60、90和120天时不同细胞的CD133+细胞比率;免疫组化观察胶质瘤干细胞的多向分化能力;用血清、无血清和干细胞3种培养液分别培养2例长期培养的胶质瘤SHG62、SH066细胞系以及对照的U87和U251细胞株,观察胶质瘤中CD133+细胞在不同培养液中的相互转换.结果:胶质瘤CD133+细胞比率随培养时间推移明显下降(P＜0.05),1周左右均降至＜1%的低水平,而细胞株则比较稳定;干细胞培养液培养的细胞中CD133+细胞比率明显高于血清和无血清培养液培养(P＜0.01);此外,胶质瘤细胞中的干细胞具有多向分化的能力,并且可以在干细胞和血清培养液中互相转换.结论:原代胶质瘤体外培养过程中CD133+细胞比率逐渐下降,而干细胞培养液能明显提高CD133+细胞比率,并且CD133+细胞可以在不同培养液中相互转换.说明生长环境对于细胞比率影响较大,提示可能存在尚未明了的影响干细胞生长分化的相关因子.     

CD133+ glioblastoma stem-like cells induce vascular mimicry in vivo

Glioblastoma is one of the most angiogenic malignancy, the neoplastic vessels of which are likely to arise by angiogenesis and vasculogenesis. An alternative mechanism of tumor vasculature is described, termed vasculogenic mimicry, by which highly aggressive tumor cells can form vessel-like structures themselves, by virtue of their high cellular plasticity. Evidence suggests that cancer stem cells acquire a multi-potent plastic phenotype and show vasculogenic potential. In this study, we report that glioblastoma stem-like cells (GSCs) can form vasculogenic mimicry in tumor xenografts and express pro-vascular molecules. We isolated GSCs from resected human glioblastoma tissues and demonstrated their stemness, differentiation, and in vivo tumor-initiating potential. Through a limiting dilution assay, CD133+ (CD133(+)-GSC) and CD133- (CD133(-)-GSC) subpopulation of GSCs were obtained. Orthotopic xenotransplantation study revealed that these two subpopulations of GSCs shared similar efficacy in tumor formation but showed distinct intratumor vasculature. In comparison with CD133(-)-GSC, a highly vascularized anaplastic tumor, mimicking vasculogenic mimicry, was found in CD133(+)-GSC-derived tumor xenografts. Subsets of CD133(+)-GSC but not CD133(-)-GSC were capable of vascular smooth muscle-like cell differentiation, in vitro and in vivo. In tumor xenografts, endothelium-associated CD31 gene was detected in implanted CD133(-)-GSC and exclusively dispersed within the tumor tissues. Although the detailed action mechanisms required further investigation, this study demonstrated the vasculogenic capacity of brain GSCs and their cellular plasticity. The results of expression of pro-vascular molecules and differentiation of vascular-like cells suggest that GSCs may contribute to form vessel-like structures and provide a blood supply for glioblastoma cells.     

Presence of pluripotent CD133+ cells correlates with malignancy of gliomas

BackgroundPresence of CD133⁺ cancer stem cells has been demonstrated within glioblastoma multiforme (GBM), the most malignant phenotype of gliomas (WHO grade IV). Since GBM frequently develops from low grade gliomas (WHO grade II) we assessed a possible qualitative or quantitative correlation of CD133⁺ cells and glioma grade to get new insights in gliomagenesis.ResultsThe amount of CD133⁺ cells within the bulk tumor mass, analyzed by immunostaining and Western blotting, showed a clear quantitative correlation with glioma grade (WHO° II, III and IV). Most of CD133⁺ cells were arranged in clusters frequently associated to tumor vessels. Protein analysis revealed high cellular coexpression of CD133 with Musashi-I but not CD34 indicating a neural, i.e. local origin of these cells. In vitro, no differences in stem cell properties concerning self-renewal and multi-lineage differentiation have been found for CD133⁺ cells isolated from gliomas of different grades.ConclusionsThese findings indicate a solely quantitative correlation of glioma grade with the presence of neural CD133⁺ cells within tumors supporting the concept of a CD133⁺ stem cell dependent gliomagenesis.     

In vitro characterization of a human neural progenitor cell coexpressing SSEA4 and CD133

The stage-specific embryonic antigen 4 (SSEA4) is commonly used as a cell surface marker to identify the pluripotent human embryonic stem (ES) cells. Immunohistochemistry on human embryonic central nervous system revealed that SSEA4 is detectable in the early neuroepithelium, and its expression decreases as development proceeds. Flow cytometry analysis of forebrain-derived cells demonstrated that the SSEA4-expressing cells are enriched in the neural stem/progenitor cell fraction (CD133(+)), but are rarely codetected with the neural stem cell (NSC) marker CD15. Using a sphere-forming assay, we showed that both subfractions CD133(+)/SSEA4(+) and CD133(+)/CD15(+) isolated from the embryonic forebrain are enriched in neurosphere-initiating cells. In addition CD133, SSEA4, and CD15 expression is sustained in the expanded neurosphere cells and also mark subfractions of neurosphere-initiating cells. Therefore, we propose that SSEA4 associated with CD133 can be used for both the positive selection and the enrichment of neural stem/progenitor cells from human embryonic forebrain.     

Type 1 collagen as a potential niche component for CD133‐positive glioblastoma cells

Cancer stem cells are thought to be closely related to tumor progression and recurrence, making them attractive therapeutic targets. Stem cells of various tissues exist within niches maintaining their stemness. Glioblastoma stem cells (GSCs) are located at tumor capillaries and the perivascular niche, which are considered to have an important role in maintaining GSCs. There were some extracellular matrices (ECM) on the perivascular connective tissue, including type 1 collagen. We here evaluated whether type 1 collagen has a potential niche for GSCs. Imunohistochemical staining of type 1 collagen and CD133, one of the GSCs markers, on glioblastoma (GBM) tissues showed CD133‐positive cells were located in immediate proximity to type 1 collagen around tumor vessels. We cultured human GBM cell lines, U87MG and GBM cells obtained from fresh surgical tissues, T472 and T555, with serum‐containing medium (SCM) or serum‐free medium with some growth factors (SFM) and in non‐coated (Non‐coat) or type 1 collagen‐coated plates (Col). The RNA expression levels of CD133 and Nestin as stem cell markers in each condition were examined. The Col condition not only with SFM but SCM made GBM cells more enhanced in RNA expression of CD133, compared to Non‐coat/SCM. Semi‐quantitative measurement of CD133‐positive cells by immunocytochemistry showed a statistically significant increase of CD133‐positive cells in Col/SFM. In addition, T472 cell line cultured in the Col/SFM had capabilities of sphere formation and tumorigenesis. Type 1 collagen was found in the perivascular area and showed a possibility to maintain GSCs. These findings suggest that type 1 collagen could be one important niche component for CD133‐positive GSCs and maintain GSCs in adherent culture.     

Role of Exosomes Derived from miR-133b Modified MSCs in an Experimental Rat Model of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) has poor outcomes due to high mortality and morbidity, but until now, the effective treatments remain limited. MicroRNAs (miRNAs) are vital regulators of gene expression and demonstrated to be linked to the pathogenesis of various central nervous system (CNS) diseases. Exosomes are considered as cell-to-cell communication vectors and secreted largely by mesenchymal stromal cells (MSCs). The present study investigated the role of miR-133b delivered by exosomes secreted from MSCs to brain tissues in rats after ICH. An autologous arterial blood ICH model in adult male Sprague–Dawley (SD) rats was used in this study. At 72 h after transfection with miR-133b mimics in MSCs, miR-133b-modified MSC-derived exosomes were collected from medium of MSCs and then injected to rats via tail vein. The levels of miR-133b in secreted exosomes and brain tissues of rats in various groups and the levels of RhoA, phosphorylations of extracellular signal regulating kinase (ERK1/2), and cAMP response element-binding protein (CREB) were detected by real-time PCR and western blot analysis, respectively. The effects of miR-133b on neuronal apoptosis and degeneration were respectively evaluated by TUNEL and fluoro-jade B staining. The miR-133b levels were reduced in brain tissues of rats at 24 h and peaked at 72 h after ICH. At 24 h after miR-133b-modified exosome administration, the level of miR-133b was significantly increased, while the apoptotic and neurodegenerative neurons were obviously reduced in brain tissues after ICH. The results of western blot analysis showed that miR-133b modified exosomes treatment remarkably suppressed RhoA expression and activated ERK1/2/CREB in brain tissues after ICH. Collectively, our investigation suggested that exosomes derived from miR-133b modified MSCs exhibited neuroprotective role for anti-apoptotic effect of miR-133b mediating RhoA and ERK1/2/CREB in rats after ICH.     

Expression of neural stem cell surface marker CD133 in balloon cells of human focal cortical dysplasia

PURPOSE: Focal cortical dysplasia (CD) is characterized by the presence of dysmorphic neurons, laminar and columnar disorganization. A few patients with CD have balloon cells intermixed with dysmorphic neurons. The cellular characteristics of balloon cells remain unknown. This study was intended to determine further the cellular characteristics of balloon cells. METHODS: Neocortical tissue resected from five patients with medically intractable focal epilepsy due to CD was studied. The presence of balloon cells (large opalescent cells with eccentric nuclei) was confirmed in all five patients by using cresylecht violet staining. Immunocytochemistry used antibodies against markers of pluripotential stem cells (CD133), multipotential progenitor cells (nestin), antiapoptotic gene products (Bcl-2), immature neurons (beta-tubulin 3, TUJ1), immature glia (vimentin), mature neurons (MAP2 and NeuN), and astrocytes (glial fibrillary acidic protein; GFAP). RESULTS: Balloon cells (BCs) were found to be immunoreactive to Bcl-2 (46%), vimentin (41%), Nestin (28%), CD133 (28%), MAP2 (27%), GFAP (14%), and TUJ1 (10%). An extremely small number of BCs were immunopositive for NeuN. Confocal double labeling showed that balloon cells were dually immunopositive for CD133/nestin; CD133/GFAP; CD133/Bcl-2, and nestin/GFAP. CONCLUSIONS: These results show that balloon cells are heterogeneous cell populations expressing cell-surface markers for pluripotential stem cells and proteins for multipotent progenitors, or immature neurons/glia. The presence of stem cell/progenitor markers in the balloon cells could be due to a persistent postnatal neurogenesis or early embryonic insult that resulted in arrest of proliferation/differentiation at their early stages. Additionally, the coexpression of Bcl-2 in CD133-positive balloon cells suggests that a resistance to programmed cell death may be involved in the pathogenesis of cortical dysplasia.     

CD133基因表达与人脑胶质瘤恶性程度相关性分析

目的探讨人脑胶质瘤组织中胶质瘤干细胞标志物CD133的表达与肿瘤恶性程度的关系。方法应用实时荧光定量PCR方法检测75例不同病理级别胶质瘤组织及4例正常脑组织中CD133基因的表达情况，并与肿瘤病理级别进行相关性分析；同时采用免疫组化法检测35例肿瘤组织和2例正常脑组织中CD133的表达情况，在蛋白表达水平予以验证。结果CD133在基因转录水平和蛋白表达水平具有良好的相关性。CD133在正常脑组织中未见表达，在各级别胶质瘤中均有表达，且差异有显著性（P〈0．01）；CD133表达量与肿瘤的恶性程度呈正相关（P〈0．01）。结论检测胶质瘤CD133表达水平有助于评价肿瘤的生物学行为，并为针对肿瘤干细胞的靶向治疗提供参考依据。     

CD133鉴定胶质瘤干细胞血管周围壁龛的实验研究

目的 检测脑肿瘤干细胞(BTSC)标记物CD133、巢蛋白(Nestin)和增殖细胞核抗原(PCNA)在74例脑胶质瘤标本中的表达,探讨肿瘤干细胞生存的微环境一壁龛的组成、形态及其在脑肿瘤组织中的分布. 方法 选取安徽医科大学附属省立医院神经外科自2007年1月至2008年10月间手术切除的74例胶质瘤标本,按照WH02000年的神经系统肿瘤分类分级标准分为Ⅱ级22例(低级别组)、Ⅲ级27例和Ⅳ级25例(高级别组),采用免疫组织化学染色和免疫荧光双标法分别检测标本中CD133的表达及其与Nestin、PCNA的共表达情况.计算并比较不同级别胶质瘤组织CD133+细胞、CD133+血管和CD133+壁龛所占的百分比.并对CD133+血管和CD133+壁龛的表达进行相关性分析. 结果 CD133+细胞聚集于壁龛内生长,低级别组胶质瘤中CD133+壁龛阳性率较低.壁龛内增殖细胞较少,与相邻肇龛之间界限清晰,周围CD133+血管分布较少.高级别组胶质瘤中CD133+壁龛阳性率高,壁龛之间无明显界限,壁龛内细胞增殖活跃,周围可见丰富的CD133+血管分布:壁龛中除CD133+/Nestin+BTSC外,可见CD133+/Nestin-细胞、CD133/PCNA+细胞等亚群细胞;不同级别胶质瘤CD133+细胞、CD133+血管、CD133+壁龛百分比不同,且肿瘤级别越高,三者表达越高,差异有统计学意义(P<0.05).CD133+壁龛与CD133+血管的表达呈正相关(r=0.425,P=0.000). 结论 在脑胶质瘤组织中存在着由CD133+/Nestin+BTSC和一些亚群细胞组成的壁龛结构,CD133+血管对于壁龛结构的维持起着非常重要的作用.     

CD44 expression in primary and recurrent oligodendrogliomas and in adjacent gliotic brain tissue

CD44 expression was evaluated in 114 primary and recurrent oligodendrogliomas (46 primary oligodendrogliomas grade II and 15 recurrences grade II; 17 primary anaplastic oligodendrogliomas and nine recurrent oligodendrogliomas grade III; 14 glioblastomas with oligodendroglial growth pattern and 13 tumour recurrences grade IV). CD44 expression was found to correlate with tumour grading (P<0.001) and with survival (Kaplan-Meier Log Rank P<0.01, median survival 28 months in oligodendrogliomas with CD44 expression vs. 108 months in CD44-negative tumours). However, multivariate Cox regression analysis of grading and CD44 expression revealed that CD44 expression had no prognostic relevance independent of histological grading. Characterization of CD44 positive cells by double labelling with GFAP revealed that in addition to oligodendroglioma cells, reactive astrocytes within the tumour, at the invasive margin and along the pathways of oligodendroglioma invasion in the subpial matrix, and in the vicinity of vessels, frequently expressed CD44. It is suggested that in analogy to carcinoma invasion where a tumour-induced production of hyaluronan was found in fibroblasts at the invasive margin of the tumour, in the brain reactive astrocytes may produce hyaluronan which would facilitate the adhesion of new CD44-positive astrocytic processes but which would also promote tumour invasion.     

胶质瘤干祖细胞SU2分化抑制、侵袭性与miRNA-125b和MMP-9关系的研究

目的 探讨胶质瘤干祖细胞SU2分化抑制、侵袭性与基质金属蛋白酶(MMP-9)和miRNA-125b的关系.方法 胶质瘤干祖细胞SU2经丙戊酸钠(sodium valproate,VPA)诱导分化得到SU2分化细胞.U87胶质瘤细胞经无血清、含生长因子的培养基培养得到U87胶质瘤干祖细胞.免疫荧光双标记法检测SU2及其分化细胞中CD133、Nestin、胶质纤维酸性蛋白( GFAP)、β微管蛋白([β-tubulinⅢ)的表达.Transwell实验检测SU2和U87胶质瘤干祖细胞的侵袭情况.实时荧光定量PCR检测SU2及U87胶质瘤干祖细胞中miRNA -125b和MMP-9基因表达.结果 SU2及其分化细胞均表达β-tubulinⅢ和GFAP.SU2细胞侵袭数明显多于U87细胞(P=0.026 1).SU2细胞中的miRNA- 125b和MMP-9表达明显高于U87细胞(均P ＜0.05).结论胶质瘤干祖细胞SU2分化抑制和高侵袭的生物学特征与miRNA- 125b和MMP-9的高表达有关,MMP-9可作为miRNA- 125b调控的靶基因进一步研究.     

Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

microRNAs(miRNAs) play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124(miR-124) overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs in bone marrow-derived mesenchymal stem cells, neural stem cells and neurons. miR-124 expression was substantially reduced in bone marrow-derived mesenchymal stem cells compared with the other cell types. We constructed a lentiviral vector overexpressing miR-124 and transfected it into bone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markers β-III tubulin and microtubule-associated protein-2 were significantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These results suggest that miR-124 plays an important role in the differentiation of bone marrow-derived mesenchymal stem cells into neurons. Our findings should facilitate the development of novel strategies for enhancing the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.