FUBP1基因在脑胶质瘤中的表达及其对U251细胞凋亡的影响

目的：研究FUBP1基因在脑胶质瘤中的表达及其对胶质瘤U251细胞凋亡的影响。方法：Real-time PCR检测35例脑胶质瘤及相应癌旁组织中FUBP1 mRNA的表达。设计并合成FUBP1基因特异性的siRNA,转染U251细胞。转染后,western blot检测转染后FUBP1蛋白的表达,双标流式细胞法检测细胞凋亡率。结果：与癌旁组织相比,FUBP1 mRNA在脑胶质瘤组织中表达明显上调。转染后,U251细胞中的FUBP1蛋白表达明显降低,同时,凋亡率明显增加。结论：FUBP1基因在脑胶质瘤中存在表达异常,其表达降低能够促进细胞凋亡。     

FUBP1基因在脑胶质瘤中的表达及其对U251细胞凋亡的影响

目的:研究FUBP1基因在脑胶质瘤中的表达及其对胶质瘤U251细胞凋亡的影响。方法:Real-time PCR检测35例脑胶质瘤及相应癌旁组织中FUBP1 mRNA的表达。设计并合成FUBP1基因特异性的siRNA,转染U251细胞。转染后,western blot检测转染后FUBP1蛋白的表达,双标流式细胞法检测细胞凋亡率。结果:与癌旁组织相比,FUBP1 mRNA在脑胶质瘤组织中表达明显上调。转染后,U251细胞中的FUBP1蛋白表达明显降低,同时,凋亡率明显增加。结论:FUBP1基因在脑胶质瘤中存在表达异常,其表达降低能够促进细胞凋亡。     

微小RNA-630对U251神经胶质瘤细胞增殖和凋亡的影响

目的 探讨微小RNA-630(miR-630)对U251神经胶质瘤细胞增殖和凋亡的影响及其调控机制。方法 荧光定量PCR(qPCR)检测人脑胶质瘤组织和细胞的miR-630表达水平。采用脂质体转染法向U251细胞转染miR-630模拟物mimics(miR-630 mimics组)及阴性对照序列(miR-NC mimics组),另选取未转染细胞为空白对照(Control)组,经qPCR验证miR-630 mimics显著增强miR-630基因表达后进一步进行功能研究：CCK-8和Hoechst染色法检测细胞增殖变化,采用流式细胞术、qPCR和Western blot分别检测凋亡细胞百分率和凋亡基因表达,qPCR和双荧光素酶报告基因系统分别检测20S蛋白酶体α1亚基(PSMA1)的表达及其与miR-630靶向关系验证。结果 miR-630在胶质瘤组织和细胞表达下调。与Control组和miR-NC mimics组相比,miR-630 mimics组U251细胞增殖能力下降,细胞凋亡增加,抗凋亡基因Bcl-2表达降低,cleaved caspase-3表达增加。过表达miR-630显著降低...     

EZH2基因沉默对人脑胶质瘤U251细胞增殖和凋亡影响的研究

目的:研究RNA干扰技术抑制zeste基因增强子人类同源物2(EZH2)的表达对人胶质瘤U251细胞增殖及凋亡的影响.方法:构建靶向EZH2基因的shRNA质粒并转染至U251细胞中,采用RT-PCR和蛋白质印迹法检测EZH2 mRNA和蛋白的表达情况,利用四甲基偶氮哇盐(MTT)实验和Annexin V-FITC/PI流式细胞术实验观察转染后U251细胞增殖和凋亡情况.结果:靶向EZH2基因的shRNA质粒成功抑制了U251细胞EZH2基因的表达,mRNA和蛋白表达抑制率分别为56.00％和88.73％;转染shRNA EZH2后,U251细胞的生长受到明显抑制(P＜0.01),在转染96 h后,生长抑制率达35.79％;转染48 h后,U251细胞早、晚期凋亡率分别为(26.59±0.83)％和(38.63±0.80)％,较阴性质粒组和空白对照组均增加,以晚期明显,差异有统计学意义,P＜0.01.结论:EZH2基因的沉默能有效抑制胶质瘤U251细胞的增殖、促进其凋亡,提示EZH2可能成为胶质瘤基因治疗的新靶点.     

BML-210对脑胶质瘤U251细胞增殖、凋亡及细胞周期的影响

目的 探讨组蛋白去乙酰化酶抑制剂BML-210 对脑胶质瘤U251细胞增殖、凋亡及细胞周期的影响。方法 采用0、5、10、20 μmol／L BML-210 处理U251细胞,活细胞计数试剂盒（CCK-8）法检测不同浓度BML-210处理24、48、72和96 h的增殖抑制率,磷酯酰丝氨酸结合蛋白-异硫氢酸荧光素／碘化丙啶双染法（Annexin-FITC／PI）双染法检测不同浓度BML-210处理后的细胞凋亡率,流式细胞仪检测不同浓度BML-210处理48 h后的细胞周期分布情况,免疫印迹检测不同浓度BML-210处理48 h后凋亡相关基因（Bcl-2、Bax和Cleaved caspase-3）蛋白表达。结果 BML-210 可呈剂量和时间依赖的方式抑制U251细胞的增殖（P＜0.05）;除5 μmol／L组的晚期凋亡率外,BML-210处理组的早、晚期凋亡率均高于对照组（P＜0.05）,且BML-210处理组作用48 h后的凋亡率均高于24 h,组间差异均有统计学意义（P＜0.05）;与对照组比较,BML-210作用48 h后U251细胞的Bcl-2水平及S、G2／M期细胞比例降低,Bax和Cleaved caspase-3水平及G0／G1期细胞比例升高,差异均有统计学意义（P＜0.05）。结论 BML-210 可抑制脑胶质瘤U251细胞的增殖并诱导其凋亡和细胞周期阻滞,对脑胶质瘤的辅助治疗有一定价值。     

AA861 对胶质瘤U251细胞增殖及凋亡的影响

目的:探讨5-脂氧合酶抑制剂AA861对胶质瘤U251细胞增殖及凋亡的影响.方法:用含有不同浓度(25μmol/L、50μmol/L、100μmol/L)AA861的培养液培养胶质瘤U251细胞,绘制细胞生长曲线;倒置相差显微镜观察细胞形态学变化;MTT法检测细胞抑制率;AO/EB荧光染色测定细胞凋亡率;Caspase-3活性检测试剂盒检测AA861干预后细胞内Caspase-3活性变化.结果:生长曲线及MTT法显示AA861对U251细胞增殖有明显的抑制作用,呈剂量和时间依赖效应;在倒置相差显微镜下,AA861诱导细胞发生凋亡形态学变化;AO/EB荧光染色显示细胞凋亡率在不同时间点及不同浓度下差异有统计学意义(P＜0.05),呈剂量和时间依赖效应;Caspase-3活性在不同时间点及不同浓度下差异有统计学意义(P＜0.05),呈剂量和时间依赖效应.结论:AA861能够抑制U251细胞的增殖,并通过Caspase-3途径诱导细胞凋亡.     

RNAi抑制PIAS3表达对胶质瘤U251细胞增殖和凋亡的影响

  目的   构建针对PIAS3的RNAi质粒载体,初步探讨抑制PIAS3表达对胶质瘤U251细胞增殖和凋亡的影响。   方法   目的:方法:构建3个RNAi载体,用脂质体法将其转染胶质瘤细胞系CHG-5,通过半定量RT-PCR筛选干扰效率最高的重组RNAi质粒。将该RNAi质粒转染体外培养的人胶质瘤U251细胞株,用半定量RT-PCR和Western blot检测PIAS3的表达。Annexin V2 FITC和PI双染流式细胞术检测细胞凋亡,流式细胞术检测细胞增殖周期变化。   结果   转染干扰质粒的细胞株PIAS3基因和蛋白表达均有减弱。流式细胞仪分析,抑制PIAS3表达能够诱导胶质瘤U251细胞拮抗凋亡（P < 0.01）,同时出现细胞周期改变,表现为G2期细胞比例升高,S期细胞比例降低,具有显著性差异（P < 0.05）。   结论   下调PIAS3基因表达使U251阻滞在G2期,拮抗细胞凋亡。       

胶质瘤细胞ING1基因表达水平与肿瘤细胞增殖和凋亡的关系研究

目的探讨胶质瘤细胞ING1基因表达水平及其与肿瘤细胞增殖活性和凋亡程度的关系.方法采用原位杂交及免疫组织化学染色方法,分别检测71例不同级别的人胶质瘤组织ING1 mRNA及ING1、PCNA蛋白,并采用TUNEL法检测原位细胞凋亡的情况.结果ING1 mRNA的阳性表达率为94.6%(35/37),ING1蛋白的阳性表达率为90.1%(64/71),两者的表达水平呈正相关(γ=0.714,P＜0.01),均随肿瘤恶性程度的增高而降低,具有显著性差异(P＜0.05).ING1表达水平降低时伴随着凋亡程度的降低及增殖活性的增强.结论胶质瘤细胞中ING1基因的表达水平与肿瘤恶性程度密切相关,并随肿瘤恶性程度的增加而相应降低.其表达异常的改变主要是转录水平调控异常的结果,并可能通过促进细胞增殖和抑制细胞凋亡,在胶质瘤的发生、发展中发挥重要作用.     

沉默UHRF1对乳腺癌细胞增殖和转移的影响

目的 探讨UHRF1对乳腺癌MDA-MB-231细胞增殖以及侵袭的影响及其相关机制。方法 采用四甲基偶氮唑盐微量酶反应比色法(MTT)检测沉默UHRF1基因后对乳腺癌MDA-MB-231细胞活力的影响;应用克隆形成实验检测沉默UHRF1后对乳腺癌MDA-MB-231细胞存活的影响;吖啶橙-溴乙锭(AO/EB)检测沉默UHRF1后对乳腺癌MDA-MB-231细胞凋亡的影响;Caspase-3活性试剂盒检测沉默UHRF1后乳腺癌细胞Casapse-3活性的变化;Western blot法检测细胞中凋亡相关蛋白Bcl-2、Bax、Bad、p-Bad、XIAP、p53、p21^Cip1/Waf1和p16^INK4a的表达;应用Transwell实验研究沉默UHRF1对MDA-MB-231细胞侵袭能力的影响;Wound Healing实验研究沉默UHRF1后对其迁移能力的影响。结果 沉默UHRF1使乳腺癌MDA-MB-231细胞活力降低;克隆形成实验结果显示沉默UHRF1后MDA-MB-231细胞存活能力降低,AO/EB染色显示沉默UHRF1促进MDA-MB-231细胞凋亡。同时Caspase-3活性实验结果显示沉默UHRF1后乳腺癌MDA-MB-231细胞Caspase-3的活性增加;Western blot结果显示,沉默UHRF1后,能够使凋亡蛋白Bad、XIAP和Bax的表达上调,同时抗凋亡蛋白p-Bad,Bcl-2的表达下调,也使p53,p21Cip1/Waf1,p16^INK4a蛋白表达升高;Transwell以及Wound Healing实验证明沉默UHRF1能够抑制乳腺癌MDA-MB-231细胞侵袭和迁移。结论 沉默UHRF1能够抑制乳腺癌MDA-MB-231细胞活力和存活,并抑制乳腺癌MDA-MB-231的侵袭和迁移。沉默UHRF1通过调控p53,p21Cip1/Waf1,p16^INK4a信号发挥作用。     

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。     

c-Met-targeted RNA interference inhibits growth and metastasis of glioma U251 cells in vitro

This phase II study was designed to determine the objective response rate and 6-month progression free survival of adult patients with recurrent supratentorial anaplastic glioma when treated with the immune modulator, polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly-ICLC). This was an open-labeled, single arm phase II study. Patients were treated with poly-ICLC alone. Patients may have had treatment for no more than two prior relapses. Treatment with poly-ICLC continued until tumor progression. Fifty five patients were enrolled in the study. Ten were ineligible after central review of pathology. Eleven percent of patients (5 of 45) had a radiographic response. Time to progression was known for 39 patients and 6 remain on treatment. The estimated 6-month progression free survival was 24%. The median survival time was 43 weeks. Poly-ICLC was well tolerated, but there was no improvement in 6-month progression free survival compared to historical database nor was there an encouraging objective radiographic response rate. Based on this study, poly-ICLC does not improve 6moPFS in patients with recurrent anaplastic gliomas but may be worth further study in combination with agents such as temozolomide.     

碱性成纤维细胞生长因子小分子干扰RNA对胶质瘤细胞株U251增殖与凋亡的影响

背景与目的:碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)是成纤维细胞生长因子(fibroblast growth factors,FGFs)家族中的重要一员,bFGF是多功能细胞因子,参与创伤愈合、组织修复、未成熟神经细胞的增殖和分化过程.本课题组前期对bFGF在胶质瘤中的表达做过研究,证实bFGF在胶质瘤细胞中过表达,并刺激胶质瘤细胞的增殖和新生血管生成.本研究检测bFGF小分子干扰RNA对胶质瘤细胞的增殖和凋亡的影响.方法:用化学法合成四条针对bFGF的siRNA和一条阴性对照siRNA,并用脂质体法转染胶质瘤细胞系U251;以Opti-MEM I无血清培养基培养的U251细胞作为正常对照.通过RT-PCR和免疫组化的方法检测bFGF的表达,并用流式细胞术、MTT法检测转染后U251细胞的凋亡和增殖活性的变化.结果:转染48 h后,正常对照、阴性对照、siRNA-1、siRNA-2、siRNA-3和siRNA-4组U251细胞中的bFGF mRNA水平分别为0.95 0.02、0.95±0.02、0.85 0.02、0.76±0.04、0.65±0.04和0.56±0.03;转染72 h后,分别为0.95±0.04、0.89±0.05、0.81±0.05、0.80±0.05、0.77±0.04和0.53±0.05.四条bFGF siRNA中,siRNA-4作用最显著.转染48 h后,siRNA-4和阴性对照组细胞增殖抑制率分别为(66.4±1.2)%和(56.3±1.4)%;转染72 h后,分别为(40.0±2.6)%和(14.7±0.6)%,siRNA-4与阴性对照组相比差异具有统计学意义(P<0.05).结论:理想的bFGF小分子干扰RNA 能够抑制该基因的表达并抑制胶质瘤细胞的增殖活性.     

Knockdown of GluR1 expression by RNA interference inhibits glioma proliferation

High-grade gliomas release excitotoxic concentrations of glutamate which contributes to their malignant phenotype. To improve our understanding of the mechanisms by which glutamate enhances tumor growth and invasion, we examined α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-mediated signaling in glioma cell lines. shRNA was used to stably knockdown GluR1, the most abundant AMPA receptor subunit in glioma, to evaluate its role in tumor signaling, proliferation and tumorigenicity. In a tissue array, there was a statistically significant increase in GluR1 expression in glioblastoma samples compared to anaplastic astrocytoma and low-grade tumors. In vitro, we observed a time and dose-dependent increase in MAPK phosphorylation following exposure to AMPA, which was blocked with AMPA receptor antagonists and the MEK1 inhibitor PD98059. Retroviral delivery of GluR1 shRNA in U251 and U87 glioma cells reduced GluR1 protein expression, inhibited AMPA-mediated increases in MAPK phosphorylation, and decreased glioma proliferation in vitro. U251 and U87 shGluR1 cells implanted into the flanks of nude mice grew slower than controls, which correlated with a decrease in proliferation measured by Ki-67 staining and an increase in apoptosis. These results suggest that AMPA receptors are abundantly expressed in high-grade gliomas and gene silencing of the GluR1 AMPA receptor subunit results in abrogation of AMPA-mediated signaling and tumor growth.     

The effect of antisense epidermal growth factor receptor (EGFR) RNA on the proliferation of human glioma cells and induction of cell apoptosis

Malignantgliomaisoneofthemostdevastatingdiseases.Theprognosisofpatientshasnotbeenimprovedevenwhenthecurrentlyavailablec0mbinedtherapiesareused.Astheknowledge0ftumorbi0l0gyandmoleculargeneticshasgreatlyincreasedoverthepastdecades,ithasbeenshownthattumorigenesisbasicallyresultsfromtheactivation0fprotooncogenesandinactivati0n0ftumorsuppressorgenes.Sincegenesencodinggrowthfactorsandtheirreceptorsarecloselyrelatedtooncogenes,muchattentionhasbeenfocusedontheroleofgrowthfactorsandtheirreceptorsinthep…     

MicroRNA-27a对U251胶质瘤细胞的影响

目的 探讨microRNA-27a(miR-27a)对U251胶质瘤细胞的影响。方法 采用体外瞬时转染的方法过表达或抑制U251细胞中的miR-27a,MTT方法检测细胞的增殖情况,Transwell侵袭试验测定U251胶质瘤细胞的侵袭能力,Real-time PCR方法测定U251细胞中miR-27a和PPARγ的含量。结果 抑制miR-27a可降低U251胶质瘤细胞增殖能力,减弱U251胶质瘤细胞侵袭能力,增加PPARγ的含量;过表达miR-27a促进U251胶质瘤细胞增殖能力,提高U251胶质瘤细胞侵袭能力,减少PPARγ的含量。结论 抑制miR-27a有利于降低U251胶质瘤细胞的增殖能力和侵袭能力。     

UHRF1 promotes proliferation of gastric cancer via mediating tumor suppressor gene hypermethylation

Epigenetic changes play significant roles in cancer development. UHRF1, an epigenetic regulator, has been shown to be overexpressed and to coordinate tumor suppressor gene (TSG) silencing in several cancers. In a previous study, we found that UHRF1 promoted gastric cancer (GC) invasion and metastasis. However, the role and underlying mechanism of UHRF1 in GC carcinogenesis remain largely unknown. In the present study, we investigated UHRF1 expression and function in GC proliferation and explored its downstream regulatory mechanism. The results demonstrated that UHRF1 overexpression was an independent and significant predictor of GC prognosis. Downregulation of UHRF1 suppressed GC proliferation and growth in vitro and in vivo, and UHRF1 upregulation showed opposite effects. Furthermore, downregulation of UHRF1 reactivated 7 TSGs, including CDX2, CDKN2A, RUNX3, FOXO4, PPARG, BRCA1 and PML, via promoter demethylation. These results provide insight into the GC proliferation process, and suggest that targeting UHRF1 represents a new therapeutic approach to block GC development.     

Regulation of UHRF1 by microRNA‐9 modulates colorectal cancer cell proliferation and apoptosis

The UHRF1 protein is pivotal for DNA methylation and heterochromatin formation, leading to decreased expressions of tumor suppressor genes and contributing to tumorigenesis. However, the factors that modulate UHRF1 expression in colorectal cancer (CRC) remain unclear. Here we showed that, compared with corresponding normal tissues, UHRF1 was upregulated and microRNA‐9 (miR‐9) was downregulated in CRC tissues. The expression of UHRF1 was inversely correlated with overall survival rates of patients with CRC. Overexpression of miR‐9 in CRC cell lines significantly attenuated CRC cell proliferation and promoted cell apoptosis. The expression of UHRF1 was markedly reduced in pre‐miR‐9 transfected CRC cells. Using luciferase reporter assay, we confirmed that miR‐9 was a direct upstream regulator of UHRF1. Finally, analysis of miR‐9 and UHRF1 levels in human CRC tissues revealed that expression of miR‐9 was inversely correlated with UHRF1 expression. Collectively, our results offer in vitro validation of the concept that miR‐9 could repress the expression of UHRF1, and function as a tumor‐suppressive microRNA in CRC. It may serve as a prognostic and therapeutic marker for CRC.