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鼻咽癌中EB病毒启动子Qp的突变及其功能学研究

Variation and Functional Study of Q Promotor of Epstein-Barr Virus in Nasopharyngeal Carcinoma

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【作者】 黄宇帆廖奕佶刘文举花文峰邓海霞买世娟谢丹宗永生

【Author】 HUANG Yu-fan1,LIAO Yi-ji1,LIU Wen-ju1,HUA Wen-feng1,DENG Hai-xia1,MAI Shi-juan1,XIE Dan1,ZONG Yong-sheng2 (1.Departmentof Experimental Research,2.Department of Pathology,State Key Laboratory of Oncology in Southern China ∥Cancer Center,Sun Yat-sen University,Guangzhou 510060,China)

【机构】 华南肿瘤学国家重点实验室∥中山大学肿瘤防治中心实验研究部华南肿瘤学国家重点实验室∥中山大学肿瘤防治中心病理科

【摘要】 【目的】探讨鼻咽癌细胞中启动EB病毒核抗原1(EBNA1蛋白)表达的EB病毒Q启动子(Qp)的变异特征,比较有第62225位点(g→a)和第62422位点(g→c)两个点突变的Qp与原型Qp(B95.8型)的功能学差异及其生物学意义。【方法】采用PCR的方法扩增29例鼻咽癌组织石蜡标本和14例健康成人外周血标本(总共43例)中的Qp序列,PCR产物测序后分析其突变情况,统计学分析Qp中的点突变与鼻咽癌的相关性。把突变型和原型Qp分别克隆到荧光素酶报告基因载体上,检测相对光强度(RLU),比较突变型与原型Qp启动转录的活性。使用染色质免疫共沉淀(ChIP)实验来比较突变型和原型Qp与Sp1蛋白的亲和力。【结果】通过PCR扩增和测序实验,证实Qp的突变与鼻咽癌有密切的相关性(P=0.0395,<0.05)。突变型Qp启动转录的活性明显高于原型(RLU之比约为2.5:1,P<0.05),并且突变型Qp与Sp1的亲和力较原型Qp增强约1.52倍。【结论】在鼻咽癌组织中,突变型Qp可能通过增强与Sp1亲和力的机制,使其启动转录的活性明显增强。Qp特定位点的突变在EB病毒对鼻咽上皮细胞的感染和转化过程中可能起了重要的作用。

【Abstract】 【Objective】 To investigate the variation of Q promoter (Qp) in nasopharyngeal carcinoma (NPC) cells,and to compare the existing two mutant sites [62 225 site(g→a)and 62 422 site (g→c) ] Qp in NPC cells with the Qp in B95.8 cell line in the functional and biological difference.【Methods】 The Qp sequence was amplified in the samples from 29 cases of paraffin- embedded tissues of NPC suffers and 14 cases of peripheral blood of healthy adults by polymerase chain reaction (PCR) method (totally 43 cases).The point mutations on specified sites were analyzed and statistically compared from sequencing results.The sequences of variant and prototype Qp were amplified by PCR and cloned into luciferase reporter vector (pGL3-basic),then transfected into HaCat cells respectively.The transcriptional activity was compared between variant and prototype Qp using luciferase reporter system.The DNA binding affinity of mutant and prototype Qp to Sp1 was compared through chromatin immunoprecipitation (ChIP) method since mutation of nt 62 225 located in a Sp1 binding site.【Results】 The mutation rate of Qp was significantly higher in NPC compared with healthy controls (P = 0.039 5,< 0.05),which suggested the variant Qp was closely associated with NPC.The transcription of the luciferase gene promoted by variant Qp was significant more than that of prototype Qp in transient transfection assay (2.5: 1,P < 0.05).The binding affinity of variant Qp to Sp1 was about 1.52 times higher than that of prototype Qp as determined by quantitative ChIP assay.【Conclusions】 The transcriptional activity was enhanced in variant Qp in NPC cells compared with prototype,which possibly through the higher binding affinity to Sp1.We suggest that the mutated Qp may play an important role during the EBV infection and transformation of nasopharyngeal epithelium.

【基金】 国家重点基础研究计划(973)项目(2006CB910104);国家自然科学基金(30400493)
  • 【文献出处】 中山大学学报(医学科学版) ,Journal of Sun Yat-Sen University(Medical Sciences) , 编辑部邮箱 ,2010年01期
  • 【分类号】R739.63
  • 【被引频次】1
  • 【下载频次】204
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