CAJ | 학술논문

[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62 225位点(g→a)和第62 422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.
[목적]탐토비인암세포중계동EB병독핵항원1(EBNA1단백)표체적EB병독Q계동자(Qp)적변이특정,비교유제62 225위점(g→a)화제62 422위점(g→c)량개점돌변적Qp여원형Qp(B95.8형)적공능학차이급기생물학의의.[방법] 채용PCR적방법확증29례비인암조직석사표본화14례건강성인외주혈표본(총공43례)중적Qp서렬,PCR산물측서후분석기돌변정황,통계학분석Qp중적점돌변여비인암적상관성.파돌변형화원형Qp분별극륭도형광소매보고기인재체상,검측상대광강도(RLU),비교돌변형여원형Qp계동전록적활성.사용염색질면역공침정(CHIP)실험래비교돌변형화원형Qp여Sp1단백적친화력.[결과]통과PCR확증화측서실험,증실Qp적돌변여비인암유밀절적상관성(P=0.0395,<0.05).돌변형Qp계동전록적활성명현고우원형(RLU지비약위2.5:1,P<0.05),병차돌변형Qp여Sp1적친화력교원형QP증강약1.52배.[결론]재비인암조직중,돌변형Qp가능통과증강여Sp1친화력적궤제,사기계동전록적활성명현증강.Qp특정위점적돌변재EB병독대비인상피세포적감염화전화과정중가능기료중요적작용.
[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 Spl 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.