Identification of tumor invasion-related differentially expressed genes in different grades and all-trans retinoic acid-treated astrocytoma cell lines

BACKGROUND:Although several genetic aberrations and gene expressional changes have been shown to exist in tumors and different grades of astrocytomas,as well as in normal tissues,the gene profiling and ge-netic pathways associated with malignant transformation and progression remain unclear. OBJECTIVE: To identify differentially expressed genes related to tumor invasion from various grades and all-trans retinoic acid (ATRA)-treated astrocytoma cell lines by cDNA microarray. DESIGN,TIME AND SETTING: In vitro...     

Screening of differentially expressed genes related to differentiation and proliferation by gene expression profiling of different grade astrocytoma cell lines*☆

BACKGROUND： The detection of differential gene expression in brain is possible by cDNA microarray technology, and the screening of differentially expressed genes might provide a biological basis for gene-targeted therapy for tumors. OBJECTIVE： To detect the differential expression of genes among astrocytoma SHG-44 （WHO grade Ⅳ）, CHG-5 （WHO grade Ⅱ）, and ATRA-treated SHG-44 cell lines by cDNA microarray. DESIGN： Laboratory experiments in vitro. SETTING： Department of Neurobiology, the Third Military Medical University. MATERIALS： The experiment was performed at the Department of Neurobiology in the Third Military Medical University of the Chinese PLA from January to October 2007. The SHG-44 cell line （WHO grade Ⅳ） was established by Prof. Ziwei Du, and the CHG-5 cell line （WHO grade Ⅱ） was set up by Prof. Xiuwu Bian from the Third Military Medical University of the Chinese PLA. The cDNA microarray containing 9182 known genes was prepared and provided by Dr. Yang Zhong at the City University of Hong Kong. METHODS： To screen differentially expressed genes from the gene expression profiles detected by cDNA microarray comparisons were made between CHG-5 and SHG-44 cells and between SHG-44 cells with or without treatment with 10 μmol/L ATRA. Some differentially expressed genes were selected randomly for Northern Blot analysis to confirm the results of the microarray. The determination criteria for differential gene expression were as follows. ① The ratio of Cy5 signal to Cy3 was greater than 2.0 or less than 0.5. ② The results of the triplicate microarray hybridizations showed the same trend in three experiments. ③ A gene appeared at least two times on the triplicate microarray hybridizations, and the 3^rd value did not show a contradictory trend. A normalized ratio of Cy5 intensity to Cy3 greater than 2.0 or less than 0.5 was considered to represent up-regulated or down-regulated gene expression, respectively. MAIN OUTCOME MEASURES： The identification of genes that were sim     

全反式维甲酸抑制MDM2基因在胶质瘤细胞SHG-44中的表达

目的 探讨全反式维甲酸对胶质瘤细胞SHG-44中MDM2基因表达的影响,为进一步研究脑胶质瘤的进展机制及基因治疗提供依据.方法 分别利用cDNA微阵列与Western blot技术分析在10μmol/L全反式维甲酸(all-transretinoic acid,ATRA)处理前后的胶质瘤SHG-44细胞MDM2基因和蛋白的差异表达应用免疫组化链霉菌抗生物素蛋白-过氧化酶(Streptavidin-Peroxidase,SP)法检测Ⅱ级与Ⅳ级胶质瘤标本MDM2蛋白的表达.随机选择数个差异基因进行Northern杂交实验,以验证cDNA微阵列的结果.结果 应用cDNA微阵列检测发现,MDM2基因在ATRA处理与未处理的SHG-44细胞之间表达量的比值为0.37,提示ATRA可抑制MDM2基因在SHG-44中的表达.该结果进一步得Northern杂交实验结果的支持.Western blot分析结果显示10μmol/L ATRA处理前后胶质瘤SHG-44细胞之间MDM2蛋白的相对表达量分别为21.40±0.58和14.02±0.35(t=24.728,P=0.000),提示MDM2蛋白在SHG-44中的表达受到ATRA抑制.Ⅱ级和Ⅳ级胶质瘤标本MDM2蛋白的阳性表达率分别为24.00%(6/25)和56.52%(13/23)(X2=5.298,P=0.021),MDM2蛋白的表达随胶质瘤恶性程度的增高而增加.结论 ATRA可抑制SHG-44胶质瘤细胞中MDM2基因的表达,MDM2基因的表达水平与胶质瘤的演进有关.     

Growth and radiosensitivity of irradiated human glioma cell progeny

BACKGROUND： Progenitors of the immortalized human glioma cell line, SHG-44, are significantly less sensitive to irradiation. Two hypotheses regarding the mechanism of this effect exist： several studies have suggested that there is a subgroup with different radiosensitivities in identical cell group, and the progenitors of irradiate is a adaptive response subgroup, so its radiosensitivity is descend. A second hypothesis suggests that irradiated glioma progeny have a stronger ability to repair DNA damage. This would suggest that when progeny are continuously irradiated, resistance to irradiation-induced DNA increases, and radiosensitivity decreases. OBJECTIVE： To investigate radiosensitivity and growth features after irradiation to progeny of the human glioma cell line SHG-44. DESIGN, TIME AND SETTING： A randomized, controlled experiment, which was performed at the Department of Radiology Laboratory, the First Hospital Affiliated to Soochow University, between September 2004 and January 2006. MATERIALS： The glioma cell line SHG-44 was provided by the Institute of Neuroscience, First Affiliated Hospital of Suzhou University. Propidium iodide reagent was provided by Coulter Corporation. A linear accelerator, KD-2 type, was provided by Siemens, Germany. The flow cytometer EPICS-XL was provided by Coulter Corporation. METHODS： Brain glioma SHG-44 cells were divided into four groups： SHG-44, SHG-44-2, SHG-44-6, and SHG-44-10 . The SHG-44-2, SHG-44-6, and SHG-44-10 cells were vertically irradiated with varying doses of 2, 6 and 10 Gy by a linear accelerator （6 MVX）. The cells were passaged for 15 generations and cultured in RPMI-1640 culture media. MAIN OUTCOME MEASURES： Community re-double time, mean lethal dose （D0）, extrapolation number （N）, fraction surviving fraction irradiated by 2 Gy dose （SF2）, quasi-threshold dose （Dq）, and cell cycle. RESULTS： The Population doubling time （PDT） of SHG-44-2, SHG-44-6, and SHG-44-10 cell groups was not significant （P = 0.052）. Compare     

miR-153下调FOXR2、CDK8和CDK13的表达抑制胶质母细胞瘤细胞增殖

目的 探讨miR-153对胶质母细胞瘤细胞增殖的影响。方法 体外培养胶质母细胞瘤细胞系U87、U251、SHG-44和T98G细胞,分别转染miR-153质粒（miR-153组）、空载质粒（载体组）和miR-153突变质粒（miR-153突变组）,另设置空白对照组（不转染任何质粒）。RT-PCR检测miR-153、FOXR2、CDK8和CDK13的表达,MTT法检测细胞增殖能力。结果 miR-153组U87、U251、SHG-44和T98G细胞miR-153表达水平较载体组和空白对照组显著上升（P<0.05）,细胞增殖水平较载体组和空白对照组均显著降低（P<0.05）。miR-153组U87、U251、SHG-44和T98G细胞FXOR2、CDK8和CDK13的mRNA水平较miR-153突变组、载体组和空白对照组均显著下降（P<0.05）,而后三组之间均无统计学差异（P>0.05）。结论 miR-153抑制胶质母细胞瘤细胞增殖,其机制可能与抑制FXOR2、CDK8和CDK13表达有关。     

过表达TBX2对胶质瘤细胞增殖和侵袭的影响

目的 探讨TBX2表达对胶质瘤细胞增殖、侵袭的影响。方法 采用RT-qPCR和免疫印迹法检测53例胶质瘤组织和瘤旁组织TBX2mRNA和蛋白表达水平。体外培养胶质瘤细胞（U251、U87和SHG-44）和正常星型胶质细胞（HA1800）,采用RT-qPCR和免疫印迹法检测细胞TBX2 mRNA和蛋白表达水平。构建TBX2过表达或低表达U251细胞株,分别采用WST-1法检测胶质瘤细胞增殖能力,Transwell实验检测细胞侵袭能力。结果 胶质瘤组织TBX2 mRNA和蛋白表达水平明显高于瘤旁组织（P<0.05）,而且,高级别胶质瘤TBX2表达水平显著高于低级别胶质瘤（P<0.05）。相比于人正常星型胶质细胞系HA1800,胶质瘤细胞系U251、U87、SHG-44细胞TBX2 mRNA和蛋白表达水平均明显增高（P<0.05）;而且,U251细胞TBX2表达水平显著高于U87和SHG-44细胞（P<0.05）,因此使用U251细胞进行后续实验。过表达TBX2显著增加U251细胞增殖和侵袭能力（P<0.05）,低表达TBX2显著抑制U251细胞增殖和侵袭能力（P<0.05）。结论 胶质瘤TBX2呈高表达,与胶质瘤增殖、侵袭能力有关。     

Differential effects of 9-cis, 13-cis and all-trans retinoic acids on the neuronal differentiation of human neuroblastoma cells

A human neuroblastoma cell line IMR-32 was used as an in vitro model to examine three naturally occurring retinoic acid (RA) isomers, 9-cis (9c), 13-cis (13c) and all-trans (AT) RA, in mediating growth differentiation and neuronal differentiation. All RA isomers inhibited cellular proliferation, with 13c-RA being most effective. Cyclic AMP-responsive-element-binding-protein (CREB) was activated during RA treatment. AT-RA was a better differentiating agent in inducing the highest expression of the neurotrophic factor receptor TrkA. After prolonged RA treatment, the expression of RA receptors (RARs) was comparable for the three isomers, but retinoid X receptors (RXRs) were differentially regulated. These results imply that distinctive molecular pathways might be involved in the in vitro differentiation of neuroblastoma with different RA isomers.     

Identification of KLRC2 as a candidate marker for brain tumor-initiating cells

ABSTRACT

Objective: Brain tumor-initiating cells are characterized by their features of self-renewal, multi-lineage differentiation, and tumorigenicity. We analyzed the gene expression of brain tumor-initiating cells to identify their novel cellular markers.

Methods: We performed cDNA microarray, in silico expressed sequence tags (ESTs), RT-PCR, and q-PCR analyses.

Results: We identified 10 genes that were more highly expressed in brain tumor-initiating cells than in neural stem cells. In addition, we identified 10 other genes that were more highly expressed in brain tumor-initiating cells than in glioma cell line cells from the cDNA microarray analysis. Using the EST database, we looked to see if the 20 genes were expressed more highly in gliomas, compared with normal adult brains. Among the 20 genes, five (KLRC2, HOXB2, KCNJ2, KLRC1, and COL20A1) were expressed more than twice in glioma samples, compared with normal adult brains, and, therefore, were referred for further evaluation. RT-PCR was conducted using cDNA samples obtained from neural stem cells, normal brain tissue, fetal brain tissue, glioma cell lines, and glioma tumor-initiating cell lines. KLRC2, a transmembrane activating receptor in natural killer cells, was expressed more highly in glioma-initiating cells than in neural stem cell lines or normal adult brain tissue. The q-PCR analysis revealed that expression of KLRC2 was significantly higher in brain tumor-initiating cells compared to normal brain controls.

Conclusion: KLRC2 could be a novel cellular marker for brain tumor-initiating cells.     

九节龙皂苷诱导胶质瘤SHG-44细胞凋亡及其机制研究

目的研究九节龙皂苷对胶质瘤SHG-44细胞潜在的治疗作用及其机制。方法用四甲基偶氨唑蓝（MTT）法检测不同剂量九节龙皂苷于不同时间（6、12、24、72h）对人胶质瘤SHG-d4细胞活性的影响和细胞流式术检测SGH-44细胞调亡情况;Western—blot检测caspase-3和Bcl-2在SHG-44细胞中的表达情况。结果流式细胞仪检测显示,随着九节龙皂苷浓度的增大和时间延长,SHG-44细胞的凋亡率明显上升,Western—blot结果提示九节龙皂苷下调了凋亡抑制蛋白Bcl-2的表达并激活了凋亡蛋白caspase-3,九节龙皂苷明显抑制SHG-44细胞的生长与增殖。结论九节龙皂苷引起胶质瘤细胞大量凋亡,具有显著的抗肿瘤作用,通过调控caspase-3和Bcl-2诱导胶质瘤SHG-44细胞凋亡。     

~(125)I诱导大鼠脑内胶质瘤凋亡实验研究

目的研究125I诱导人脑胶质瘤细胞系SHG-44体内外凋亡的可能性及其机制。方法体外培养SHG-44细胞,采用流式细胞仪法检测125I诱导SHG-44细胞凋亡及对细胞周期影响,采用立体定向的方法建立大鼠脑内人胶质瘤模型,1周后经MRI检测后,于肿瘤区接种125I,2周后复查MRI检测肿瘤大小,3周后处死大鼠,取对照组及肿瘤周边组织及肿瘤组织行Bcl-2、p53免疫组织化学染色。结果SHG-44细胞接种1周,MRI示脑内形成实体瘤;125I可抑制肿瘤生长,诱导细胞凋亡,延长荷瘤鼠生长周期,抑制Bcl-2基因表达,促进p53蛋白表达。结论125I具有体内、外抑制SHG-44细胞增殖,诱导凋亡的作用;其诱导凋亡机制可能与抑制Bcl-2蛋白表达,促进p53蛋白表达有关。     

他莫昔芬对SHG-44胶质瘤细胞氯通道的抑制作用

目的研究氯通道阻断剂他莫昔芬（tamoxifen）对SHG-44胶质瘤细胞电压依赖性氯通道电流的作用。方法采用全细胞膜片钳方法记录SHG-44细胞的电压依赖性氯通道电流，并观察施用不同浓度的他莫昔芬后电流的变化。结果该电流特性为外向整流、不失活，他莫昔芬能够明显阻断该电流，该阻断具有剂量依赖性及电压依赖性。在＋100mV时，1μM及5μM他莫昔芬对氯电流抑制率分别为48％及89％。结论他莫昔芬可明显阻断SHG-44胶质瘤细胞上的电压依赖性氯通道。     

利用基因芯片研究与胶质母细胞瘤侵袭性相关的基因

目的探讨利用基因表达谱芯片筛选人脑胶质母细胞瘤与侵袭性相关基因的表达及功能。方法用含13 939种人类基因的BioStarH140S型芯片,以成人脑及6例胶质母细胞瘤组织总RNA制备的探针杂交芯片;ScanArray4 000扫描芯片荧光信号,提取脑及胶质母细胞瘤组织差异基因,并进行生物信息分析及功能研究。结果表达谱芯片筛选出胶质母细胞瘤差异基因198条(1.42％),与细胞信号和传递蛋白、细胞骨架、代谢、蛋白翻译合成、细胞周期蛋白类、癌基因和抑癌基因等多类基因密切相关;与侵袭性相关的8条细胞骨架和细胞外基质基因表达谱相似,均在胶质母细胞瘤中显著上调,生物信息分析为α-连环素基因、钙粘附素1基因、层粘连蛋白、纤连蛋白1基因、基质金属蛋白酶2、Ⅲ型胶原基因、组织金属蛋白酶抑制1基因和血小板衍生生长因子受体A基因。结论表达谱芯片是高通量筛选胶质瘤相关基因的生物高新技术,侵袭性相关基因为判断胶质母细胞瘤患者的预后提供了分子生物学指标,有助于临床诊治。     

^125I诱导大鼠脑内胶质瘤凋亡实验研究

目的 研究^125I诱导人脑胶质瘤细胞系SHG-44体内外凋亡的可能性及其机制。方法 体外培养SHG-44细胞，采用流式细胞仪法检测^125I诱导SHG-44细胞凋亡及对细胞周期影响，采用立体定向的方法建立大鼠脑内人胶质瘤模型．1周后经MRI检测后．于肿瘤区接种^125I，2周后复查MRI检测肿瘤大小，3周后处死大鼠，取对照组及肿瘤周边组织及肿瘤组织行Bcl-2、p53免疫组织化学染色。结果 SHG-44细胞接种1周，MRI示脑内形成实体瘤；^125I可抑制肿瘤生长，诱导细胞凋亡。延长荷瘤鼠生长周期。抑制Bcl-2基因表达，促进p53蛋白表达。结论^125I具有体内、外抑制SHG-44细胞增殖，诱导凋亡的作用：其诱导凋亡机制可能与抑制Bcl-2蛋白表达，促进p53蛋白表达有关。     

Gene expression profiling by mRNA sequencing reveals increased expression of immune/inflammation-related genes in the hippocampus of individuals with schizophrenia

Whole-genome expression profiling in postmortem brain tissue has recently provided insight into the pathophysiology of schizophrenia. Previous microarray and RNA-Seq studies identified several biological processes including synaptic function, mitochondrial function and immune/inflammation response as altered in the cortex of subjects with schizophrenia. Now using RNA-Seq data from the hippocampus, we have identified 144 differentially expressed genes in schizophrenia cases as compared with unaffected controls. Immune/inflammation response was the main biological process over-represented in these genes. The upregulation of several of these genes, IFITM1, IFITM2, IFITM3, APOL1 (Apolipoprotein L1), ADORA2A (adenosine receptor 2A), IGFBP4 and CD163 were validated in the schizophrenia subjects using data from the SNCID database and with quantitative RT-PCR. We identified a co-expression module associated with schizophrenia that includes the majority of differentially expressed genes related to immune/inflammation response as well as with the density of parvalbumin-containing neurons in the hippocampus. The results indicate that abnormal immune/inflammation response in the hippocampus may underlie the pathophysiology of schizophrenia and may be associated with abnormalities in the parvalbumin-containing neurons that lead to the cognitive deficits of the disease.     

白藜芦醇抑制SHG-44胶质瘤细胞生长实验研究

目的探讨白藜芦醇(Res)在体外诱导脑胶质瘤SHG-44细胞凋亡并抑制其生长的作用。方法四甲基偶氮唑蓝(MTT)比色法测量不同剂量的Res作用6h、24h和48h后对SHG-44胶质瘤细胞增殖的影响。HE染色、Hoechst33342荧光染色观察细胞形态改变,DNAladder检测细胞DNA裂解情况,流式细胞仪用异硫氰酸荧光素标记的膜联蛋白V(AnnexinV-FITC)和碘化丙啶(PI)双染检测凋亡率,并测定细胞周期的改变。结果Res明显抑制SHG-44细胞的生长和增殖(P<0.01),呈浓度及时间依赖性反应;Res所致的SHG-44胶质瘤细胞凋亡为浓度依赖关系,随着浓度的增高,凋亡更明显。此凋亡细胞周期主要发生G1期比例升高,S、G2期比例降低。结论Res明显抑制SHG-44细胞生长并诱导其发生凋亡和细胞周期改变,为Res用于治疗脑胶质细胞瘤提供了实验依据。     

脂质体介导CD基因转染SHG-44胶质瘤细胞凋亡的实验研究

目的以含胞嘧啶脱氨酶（CD）基因的pCMVCD重组表达质粒转染SHG-44胶质瘤细胞，体外观察5-氟胞嘧啶（5-FC）对转染CD基因的胶质瘤细胞的凋亡诱导作用。方法体外扩增、酶切鉴定pCMVCD质粒并采用DNA序列测定pCMVCD质粒中的CD基因；脂质体Lipofectamine 2000介导pCMVCD质粒转染SHG-44细胞，G418筛选培养获取抗性细胞克隆（即SHG-44／CD细胞）；免疫细胞化学检测SHG-44／CD细胞的CD基因蛋白水平表达；流式细胞仪、TUNEL实验及透射电子显微镜观察5-FC对表达CD基因的SHG-44／CD细胞凋亡的诱导作用。结果含CD基因的pCMVCD质粒成功转染进入SHG-44细胞，获取了含CD基因的SHG-44／CD细胞，免疫细胞化学染色显示SHG-44／CD细胞成功地表达了CD。在含5-FC的培养液中培养，SHG-44／CD细胞出现典型的凋亡形态，TUNEL显示凋亡细胞比例极高；透射电镜可见凋亡改变；流式细胞术检测凋亡率达18．6％，凋亡率呈剂量依赖性。结论建立了SHG-44恶性人脑胶质瘤细胞的CD／5-FC自杀基因系统。诱导SHG-44／CD胶质瘤细胞产生凋亡可能是脑胶质瘤CD基因疗法的重要机制。