腺相关病毒介导RNA干扰抑制EB病毒潜伏膜蛋白1基因表达

目的构建针对鼻咽癌(nasopharyngeal carcinoma,NPC)EB病毒(Epstein-Barr virus,EBV)潜伏膜蛋白1(latent membrane protein 1,LMP-1)的特异性发夹状RNA(small hairpin RNA,shRNA)干扰序列的重组腺相关病毒(recombinant adeno-associated virus,rAAV)载体(rAAV-shRNA- LMP-1),介导其体外表达并鉴定其生物活性。方法设计针对EBV-LMP-1的特异性shRNA干扰序列,采用分子克隆的方法,将干扰序列及U6启动子插入pAAV-2质粒中,采用无需包装辅助病毒的双质粒共转染方法制备rAAV-shRNA-LMP-1,测定其滴度及感染效率,RT- PCR鉴定目的基因的抑制效率。结果以rAAV-EGFP 5×10~4v．g(Virus genome,病毒基因组数),细胞转染C666-1细胞,转染效率大于95%,PT-PCR鉴定rAAV- shRNA-LMP-1以5×10~4v．g/细胞转染C666-1后目的基因抑制效率大于90%。结论通过rAAV介导RNA干扰能有效抑制LMP-1基因表达,为进一步研究肿瘤基因治疗,尤其是动物实验奠定了基础。     

EB病毒潜伏膜蛋白1对鼻咽癌细胞体外转移能力影响

目的探讨EB病毒(epstein-barr virus,EBV)潜伏膜蛋白1(Iatent memb rane protein 1,LMP-1)对鼻咽癌(nasopha ryngeal carcinoma,NPC)细胞体外转移能力的影响。方法设计针对EBV-LMP-1的特异性发夹状RNA(small hairpin RNA,shRNA)干扰序列,构建2种重组腺相关病毒(recombinant adeno-associated virus,rAAV)载体:rAAV-增强型绿色荧光蛋白(enhance green fluorescent protein,EGFP)和rAAV-shRNA-LMP-1,以不同滴度rAAV-EGFP转染鼻咽癌C666-1细胞确定最佳转染效率(multiplicity of infection,MOI),rAAV-shRNA-LMP-1按MOI转染C666-1,RT-PCR鉴定抑制效率,划痕试验和基底膜穿透试验检验细胞转移能力的变化。结果以rAAV-EGFP 5×10~4v.g(virus genome,病毒基因组数)/细胞转染C666-1细胞,转染效率大于95%,RT-PCR鉴定rAAV-shRNA-LMP-1以5×10~4v.g/细胞转染C666-1后目的基因抑制效率大于90%,划痕试验显示在相同的时间内,越过划痕边缘移动到空白处的细胞数明显减少(P<0.01),基底膜穿透试验提示细胞穿透能力显著下降(P<0.001)。结论通过rAAV介导RNA干扰能有效抑制LMP-1基因表达,并显著抑制了肿瘤细胞的运动及穿透转移能力。     

沉默变异性浆细胞瘤异位１抑制鼻咽癌细胞增殖作用的机制研究

目的探讨变异性浆细胞瘤异位１（ｐｌａｓｍａｃｙｔｏ ｍａｖａｒｉａｎｔｔｒａｎｓｌｏｃａｔｉｏｎ１,ＰＶＴ１）抑制鼻咽癌细胞增殖的分子机制。方法构建特异性的shRNA PVT1慢病毒载体,包装慢病毒（LV/shRNA PVT1）并转导鼻咽癌C666 1细胞,检测Smad4的表达;LV/shRNA PVT1转导C666 1细胞24 h后转染Smad4 siRNA,通过细胞计数、四甲基偶氮唑蓝（MTT）法及流式细胞术分析沉默Smad4对LV/shRNA PVT1介导的鼻咽癌细胞生长及细胞周期的影响,并检测细胞周期相关调控因子的表达。结果下调PVT1后鼻咽癌细胞中Smad4的表达升高,而用siRNA沉默Smad4表达后,结果显示,抑制Smad4表达可阻断LV/shRNA PVT1介导的细胞生长抑制效应,同时细胞周期结果显示,与对照组相比,shRNA PVT1/Smad4 siRNA组细胞G1期比例降低,S期和G2期比例升高,并且CDK4、CDK6及c myc的mRNA水平升高。结论下调PVT1可抑制鼻咽癌细胞增殖,并升高Smad4的表达,沉默Smad4可阻断shRNA PVT1对鼻咽癌细胞增殖的抑制效应;提示Smad4可能是PVT1作用的一个下游靶基因,下调PVT1可能通过Smad4抑制鼻咽癌细胞增殖。     

RNA干扰细胞周期蛋白D1对Hep-2周期和凋亡的影响

目的研究小干扰RNA(small interferingRNA,siRNA)抑制细胞周期蛋白D1(cyclin D1)对喉鳞状细胞癌(简称喉癌)细胞生长和凋亡的影响。方法通过脂质体转染试剂将针对cyclin D1的特异小干扰RNA转入喉癌细胞,利用MTT法检测喉癌细胞生长抑制情况,流式细胞技术检测喉癌细胞周期变化和细胞凋亡。结果MTT结果表明喉癌细胞生长受到抑制,并具有时间依赖性。同时实验组喉癌细胞周期受到影响,并检测到喉癌细胞的凋亡。结论Cyclin D1基因沉默后,喉癌细胞生长受到了抑制,细胞周期受到影响,并能够最终导致喉癌细胞凋亡。     

RNA干扰与鼻咽癌的研究进展

RNA干扰（RNAi）是利用双链小片段RNA高效、特异性地降解细胞内同源mRNA,阻断体内基因表达,使细胞出现靶基因缺失的表型［1］。近几年,RNA干扰研究在国内外迅速开展,并且扩大到许多方面,如抑制病毒的研究、抑制多药耐药基因的表达 、在人体寄生虫研究中的应用、基因治疗、肿瘤研究等。而鼻咽癌的发病机制与许多致病因素有关,实验研究表明RNAi可以通过多种途径影响鼻咽癌细胞的生长,因此RNAi可能为鼻咽癌治疗提供新的方法。     

利用RNA干扰效应阻抑鼻咽癌细胞bcl-xL基因表达和诱导癌细胞凋亡的研究

目的研究RNA干扰(RNA interference)效应对人鼻咽癌低分化上皮细胞株CNE-2Zbcl-xL基因表达的抑制作用及其对CNE-2Z细胞增殖抑制和凋亡诱导的作用。方法使用美国Ambion公司提供的网上设计软件设计针对人bcl—xL基因的小干扰RNA(siRNA)序列，体外转录试剂盒合成siRNA；荧光素标记试剂盒标记siRNA；脂质体法将siRNA转入CNE-2Z细胞株。荧光显微镜下观察siRNA的转染效率；RT—PCR法半定量检测siRNA对bcl—xL基因表达的抑制作用；噻唑蓝法检测siRNA对细胞生长增殖抑制作用；流式细胞术检测siRNA诱导细胞凋亡作用。结果荧光显微镜下荧光素标记的siRNA转染组可见到细胞内清晰的绿色荧光，而在未转染siRNA对照组未见；各siRNA转染组bcl—xL mRNA表达水平有不同程度的下调，下调范围在10％～70％之间，而在未转染对照组内bcl—xLmRNA表达水平无明显改变；细胞生长增殖抑制率在一定范围内具有剂量依赖性(剂量增加，抑制率增高)和时间依赖性；各浓度siRNA4转染组可不同程度诱导CNE-2Z细胞凋亡。结论体外转录合成的siRNA能特异有效地下调bcl—xL基因的表达，不同序列特异性的siRNA下调bcl—xL基因表达的能力不同；瞬时转染bcl—xLsiRNA4能有效抑制鼻咽癌细胞增殖并诱导其凋亡；siRNA不仅为基因组功能分析提供了强有力的工具，而且为抗鼻咽癌基因治疗提供了新的思路。     

RNA干扰DJ-1基因抑制喉癌细胞的增殖

目的 探讨RNA干扰(RNAi)抑制喉癌Hep-2细胞DJ-1基因的表达及观察其细胞效应.方法 设计合成3对特异性针对人DJ-1基因的小干扰RNA(siRNA),脂质体法转染Hep-2细胞,实验分为4组:瞬时转染非特异性siRNA对照组及转染特异性siRNA 3组(siRNA1、siRNA2、siRNA3).采用RT-PCR检测DJ-1 mRNA,蛋白免疫印迹法检测DJ-1蛋白,流式细胞仪检测细胞凋亡及四甲基偶氮唑蓝(MTT)比色法检测特异性siRNA1组对Hep-2细胞效应.结果 本实验设计合成的3对特异性siRNA均不同程度地抑制DJ-1基因表达,其中特异性siRNA1组抑制效果最佳.M1Tr比色法结果显示特异性siRNAl组(终浓度50 nmo~L、100 nmolZL)对Hep-2细胞增殖具有抑制作用.流式细胞仪检测结果最示特异性siRNA1组能诱导Hep-2细胞凋亡,转染特异性siRNA1组凋亡率为(15.7±4.8)%,非特异性siRNA对照组凋亡率为(4.5±0.4)%,非特异性siRNA对照组与特异性siRNA1组凋亡率比较差异有统计学意义(t=4.736,P<0.01).结论 转染特异性siRNA组能有效抑制Hep-2细胞增殖,诱导Hep-2细胞凋亡.     

Skp2 siRNA表达载体构建及其对喉鳞状细胞癌细胞Skp2基因表达的抑制作用

目的:应用RNA干扰技术,构建针对Skp2的siRNA表达载体,抑制喉鳞状细胞癌细胞Skp2基因的表达。方法:根据设计siRNA的原则,针对人Skp2的mRNA序列,设计并合成编码siRNA的2条寡核苷酸序列,经退火成互补双链,再克隆到真核表达载体pGPU6/Neo中构建重组体pGPU6Skp2,转化DH5α菌株,提取质粒进行酶切鉴定后,进行序列测定。然后转染重组质粒至Hep2细胞中,应用流式细胞术检测Skp2基因的表达。结果:将合成的DNA序列退火后克隆到载体上,经酶切和测序鉴定确实为所需序列。pGPU6Skp2转染细胞后,Skp2基因在蛋白水平的表达量受到明显抑制。结论:成功构建了针对人Skp2的siRNA表达载体,通过转染Hep2细胞,可有效抑制细胞Skp2的表达,为后续基因治疗研究奠定了实验基础。     

X射线交错互补修复基因1多态性和EB病毒感染对鼻咽癌细胞基本特性的影响

目的　探讨X射线交错互补修复基因1（X-ray repair cross-complementing gene 1,XRCC1）多态性与EB病毒感染对鼻咽癌细胞基本特性的影响。方法　将鼻咽癌细胞株分组,使之分别表达不同的XRCC1基因型,转染EB病毒,比较转染前后不同基因型鼻咽癌细胞的基本特性,并检测XRCC1蛋白的表达水平。结果　XRCC1不同基因型的鼻咽癌细胞之间基本特性无明显差异,XRCC1蛋白水平无差异。EB病毒感染后,各组鼻咽癌细胞生长加快、增殖能力变强、迁移速度加快、凋亡率降低,XRCC1蛋白水平无明显改变。EB病毒感染后,194位点突变型的鼻咽癌细胞与280、399位点突变型细胞相比,细胞增殖和迁移能力改变程度较小,差异有统计学意义。其他基因型细胞基本特性及XRCC1蛋白水平无差异。结论　EB病毒感染可增加鼻咽癌易感性,EB病毒感染后XRCC1 194位点的Trp突变型基因可能对鼻咽癌有保护作用。     

咽拭子检测潜伏膜蛋白1在鼻咽癌诊断中的应用

目的：验证咽拭子法检测EB病毒（EBV）潜伏膜蛋白1（LMP1）在鼻咽癌诊断中应用的可行性;检测LMP1野生型及30bp缺失变异型在鼻咽癌中的分布情况。方法：用咽拭子法收集鼻咽癌组及对照组鼻咽部脱落细胞,微量提取DNA,经PCR扩增人β珠蛋白基因序列验证咽拭子法的可行性,用特异性引物扩增出特异的LMP1序列,验证其在鼻咽癌诊断中的意义。测序分析LMP1变异情况。结果：咽拭子合格率为96．4％,LMP1基因作为鼻咽癌的检测指标,灵敏度为91．7％,特异性为95．6％,其中变异型LMP1在鼻咽癌中的表达频率为80．6％,野生型为11．1％。结论：咽拭子法可作为一种基因检测手段,LMP1基因的检测可以协同EB病毒壳抗原（EBVCA）-IgA作为鼻咽癌诊断的辅助指标,在LMP1（＋）的鼻咽癌患者中LMP1—30bp缺失突变普遍存在。     

Latent Membrane Protein-1 Oncogene Deletions in Nasopharyngeal Carcinoma in Caucasian Patients

Objective --The latent membrane protein-1 (LMP-1) is an Epstein-Barr virus (EBV)-transforming protein expressed in nasopharyngeal carcinoma (NPC). A 30-bp deletion in the LMP-1 oncogene has been described in NPC patients from Asia. The purpose of this study was to evaluate the association between NPC and such an EBV deletion in Caucasian patients. Material and Methods --Twenty-seven patients with a diagnosis of NPC were selected. Most of the NPCs were classified as Stages III and IV using the International Union Against Cancer system. Formalin-fixed, paraffin-embedded NPC specimens were found for these cases. Hematoxylin-eosin slides were reviewed and survival analysis was done using the log-rank method. In situ hybridization for EBV-encoded non-polyadenylated RNAs and expression of LMP-1 by means of immunohistochemistry was also performed. Polymerase chain reaction for LMP-1 oncogene analysis was performed to detect the presence of a 30-bp deletion in NPC specimens and EBV-related controls. Results --The 30-bp deletion was identified in 67% of NPC cases and in 30% of controls, a statistically significant difference (p = 0.01, χ² test). LMP-1 deletion was not statistically associated with a worse prognosis in NPC patients (5-year survival: 33% in wild-LMP-1 strains vs 24% in deleted-LMP-1 strains; p = 0.053, log-rank test). Conclusion --A 30-bp deletion in the LMP-1 oncogene is present in more than half of Caucasian NPC cases EBV carrying partial deletions in the LMP-1 oncogene may play a role in the pathogenesis of NPC in Caucasian patients.     

Latent membrane protein-1 oncogene deletions in nasopharyngeal carcinoma in Caucasian patients

OBJECTIVE: The latent membrane protein-1 (LMP-1) is an Epstein-Barr virus (EBV)-transforming protein expressed in nasopharyngeal carcinoma (NPC). A 30-bp deletion in the LMP-1 oncogene has been described in NPC patients from Asia. The purpose of this study was to evaluate the association between NPC and such an EBV deletion in Caucasian patients. MATERIAL AND METHODS: Twenty-seven patients with a diagnosis of NPC were selected. Most of the NPCs were classified as Stages III and IV using the International Union Against Cancer system. Formalin-fixed, paraffin-embedded NPC specimens were found for these cases. Hematoxylin-eosin slides were reviewed and survival analysis was done using the log-rank method. In situ hybridization for EBV-encoded non-polyadenylated RNAs and expression of LMP-1 by means of immunohistochemistry was also performed. Polymerase chain reaction for LMP-1 oncogene analysis was performed to detect the presence of a 30-bp deletion in NPC specimens and EBV-related controls RESULTS: The 30-bp deletion was identified in 67% of NPC cases and in 30% of controls, a statistically significant difference (p = 0.01, chi2 test). LMP-1 deletion was not statistically associated with a worse prognosis in NPC patients (5-year survival: 33% in wild-LMP-1 strains vs 24% in deleted-LMP-1 strains; p = 0.053, log-rank test). CONCLUSION: A 30-bp deletion in the LMP-1 oncogene is present in more than half of Caucasian NPC cases EBV carrying partial deletions in the LMP-1 oncogene may play a role in the pathogenesis of NPC in Caucasian patients.     

Overexpression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with epidermal growth factor receptor expression

OBJECTIVES: To examine the association between cyclooxygenase-2 (COX-2) expression with epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and latent membrane protein 1 (LMP-1) expression and with COX-2 promoter methylation status in primary nasopharyngeal cancer (NPC) tumors and to determine COX-2 promoter methylation status in NPC cell lines. DESIGN: Retrospective study. SETTING: Patients with NPC were referred to the Department of Otolaryngology-Head and Neck Surgery for treatment. PATIENTS: Formalin-fixed, paraffin-embedded NPC specimens from 42 patients were obtained. INTERVENTIONS: Immunohistochemical expression of COX-2, EGFR, VEGF, iNOS, and LMP-1 was performed in 42 NPC samples. COX-2 promoter methylation status was studied in 20 separate specimens and in 4 NPC cell lines. MAIN OUTCOME MEASURES: (1) COX-2, EGFR, VEGF, iNOS, and LMP-1 expression; and (2) COX-2 promotor methylation status. RESULTS: COX-2 was overexpressed in 79% of NPC specimens and was associated with EGFR status (P = .03) but not with LMP-1 or iNOS. In primary NPC tissue, methylation of the COX-2 promoter was seen in 4 of 7 COX-2-negative and 1 of 13 COX-2-positive immunohistochemical cases. COX-2 promoter methylation was found in the CNE-1 cell line. CONCLUSIONS: Nasopharyngeal cancer may be a useful target for selective COX-2 inhibition. The absence of promoter methylation may be a necessary component of COX-2 overexpression, and promoter methylation may be one of the mechanisms that regulate COX-2 expression.     

Epstein-Barr virus latent membrane protein-1 (LMP-1) expression in oral squamous cell carcinoma

OBJECTIVES: Epstein-Barr virus (EBV) is frequently associated with malignant cell transformation through the action of the oncoprotein latent membrane protein-1 (LMP-1). The present study aimed to determine the presence of EBV in oral squamous cell carcinomas (OSCCs) and the expression of LMP-1 in neoplastic cells of EBV-positive OSCCs. STUDY DESIGN/METHODS: In a retrospective study of 78 OSCCs, we investigated the presence of the DNA of EBV by polymerase chain reaction, the expression of the oncoprotein LMP-1 by immunohistochemistry, and the presence of EBV-encoded RNA (EBER) by in situ hybridization. RESULTS: EBV DNA was detected in 19.2% of the cases. Expression of LMP-1 in neoplastic cells was found in 85.7% of the EBV-positive OSCCs. EBV presence was significantly more frequent (P <.05) in OSCCs localized on the lateral tongue. EBV-positive OSCCs more frequently presented (P <.05) greater nuclear atypia. CONCLUSION: EBV can appear in latent form in OSCC and express its main oncoprotein, LMP-1.     

小发卡状RNA联合脱氧核酶抑制鼻咽癌细胞中EB病毒潜伏膜蛋白基因表达研究

目的:探讨小发卡状RNA单独及联合脱氧核酶对鼻咽癌细胞中EB病毒EBV潜伏膜蛋白1(LMP1)基因表达的抑制作用。方法:带绿色荧光报告基团的LMP1mRNA表达载体,小发卡状RNA及脱氧核酶按照阳性对照组、RNA干扰组、联合组和脱氧核酶组4组分别共转染HNE1细胞,观察分析计数细胞中绿色荧光表达情况,检测LMP1mRNA和蛋白表达水平。结果:联合组细胞绿色荧光OD值均数与RNA干扰组差异有统计学意义(P<0.01);联合组绿色荧光抑制率分别为86.31%和88.88%,比RNA干扰组抑制率75.15%高;EBVLMP1mRNA和蛋白水平检测提示联合组抑制效率较RNA干扰组高。结论:小发卡状RNA联合脱氧核酶能够提高抑制EBV-LMP1基因表达的效率。     

Monitoring tumor recurrence with nasopharyngeal swab and latent membrane protein-1 and epstein-barr nuclear antigen-1 gene detection in treated patients with nasopharyngeal carcinoma

OBJECTIVES: Epstein-Barr virus (EBV) is closely related to nasopharyngeal carcinoma (NPC). Detection of EBV genomic DNA in a nasopharyngeal swab specimen may indicate the presence of NPC, and the EBV genomic DNA is only detected in patients with NPC and not in other head and neck cancers. This study aims to prove that detection of EBV genomic DNA by means of the latent membrane protein (LMP)-1 gene and the Epstein-Barr nuclear antigen (EBNA)-1 gene in the nasopharynx in NPC patients after radiation therapy indicates local recurrence of NPC. STUDY DESIGN: Prospective. METHODS: Nasopharyngeal swab with polymerase chain reaction (PCR)-based LMP-1 and EBNA-1 gene detection was used to monitor local recurrence in 84 NPC patients who completed radiation therapy. RESULTS: Of the 12 patients demonstrating positive LMP-1 and EBNA-1 gene, 11 had local recurrence, and 10 of them had early rT1 mucosal recurrence. Subsequent salvage nasopharyngectomy controlled local disease in nine. Only one local recurrence in the skull base failed to show LMP-1 gene initially. Detection of LMP-1 gene and later verification with EBNA-1 gene from nasopharyngeal swabs in NPC patients after radiation therapy predicted local recurrence with a sensitivity of 91.7% and a specificity of 98.6%. CONCLUSIONS: Nasopharyngeal swab with LMP-1 and EBNA-1 gene detection is a useful and reliable method to monitor local recurrence in NPC patients. It helps to detect recurrence early and may improve local control and enhance survival.     

RNA同时干扰四个不同基因对鼻咽癌细胞CNE-2Z生长增殖的实验研究

目的 应用RNA干扰技术构建一个载体同时编码四个基因,即血管内皮生长因子(VEGF)、致癌基因蛋白(c-myc)、存活素(survivin)和端粒酶反转录酶的短发夹重组质粒,并与单基因质粒做对比,通过体内外实验,研究其对鼻咽癌细胞CNE-2Z生长增殖的影响.方法 构建同时表达VEGF、c-myc、survivin和端粒酶反转录酶并含荧光素标记的短发夹RNA质粒命名为P-1,并构建单独表达VEGF、c-myc、survivin和端粒酶反转录酶的质粒,分别命名为P-2、P-3、P-4和P-5,分别将其将转染人鼻咽癌CNE-2Z细胞株及荷瘤裸鼠瘤体内.以激光共聚焦显微镜观察质粒在CNE-2Z细胞及瘤体内的转染及表达情况.四甲基偶氮噻唑蓝法检测细胞增殖活性,实时PCR在mRNA水平上检测对目的基因表达的抑制作用,免疫印迹法在蛋白水平上检测对目的基因的封闭效果,Trans well侵袭小室模型观察导入CNE-2Z细胞对其恶性生物学行为的改变,流式细胞仪观察各组凋亡率,体内裸鼠成瘤实验观察质粒对肿瘤的抑制效果.结果 多基因干扰质粒转染鼻咽癌细胞后,CNE-2Z细胞增殖受到明显抑制,体外侵袭能力显著下降(P值均＜0.05).实时PCR证实多基因组4个不同基因mRNA表达均降低,免疫印迹技术验证目的基因蛋白同时表达下调.流式细胞仪结果证明多基因凋亡率明显高于单基因组(P值均＜0.05).体内抑瘤实验表明,与阴性组和空白组相比,P-1组移植瘤生长明显受到抑制(P＜0.05),并且多基因质粒抑制肿瘤细胞增长的作用要强于单基因干扰质粒(户值均＜0.05).结论 同时干扰多个基因能有效抑制鼻咽癌细胞增殖并诱导其凋亡,为鼻咽癌基因治疗奠定了良好的实验基础.