卷曲蛋白跨膜受体7基因启动子的构建及其肝癌特异性转录调控活性研究  

Construction of frizzled transmembrane receptor 7 gene promoter and analysis of its tumor-specific transcriptional regulatory activity in hepatocellular carcinoma

作　　者：许红攀 李智洋[1] 龚来玲 夏艳艳[1] 庞露[1] 司进[1] 

机构地区：[1]南京医科大学第二附属医院检验科,210011

出　　处：《中华实验外科杂志》2016年第4期965-968,共4页Chinese Journal of Experimental Surgery

基　　金：基金项目：国家自然科学基金（81472831）;江苏省科教兴卫工程项目（RC2011081）;江苏省“六大人才高峰”（2013-WSN-054）

摘　　要：目的构建卷曲蛋白跨膜受体7（FZD7）基因启动子，并分析其肝癌特异性转录调控活性。方法构建FZD7启动子及FZD7启动子变异体（MutFZD7），并设计合成荧光报告载体pFZD7-绿色荧光蛋白（GFP）、MutpFZD7-GFP。将各重组质粒载体转染至肝癌细胞HepG2、SMMC7721及正常肝细胞1．02中，用荧光显微镜和流式细胞仪检测GFP的表达。在裸鼠肝癌移植瘤模型瘤体内注射各重组质粒后，制备肿瘤组织冰冻切片并在荧光显微镜下观察GFP表达。结果转染pFZD7-GFP的肝癌细胞HepG2和SMMC7721中均有高强度荧光，且GFP表达率分别为（45．1±3．1）％和（25．3±2．5）％，显著高于L02细胞的GFP表达率[（0．4±0．3）％，P〈0．05]；而转染MutpFZD7-GFP的HepG2、SMMC7721和L02细胞中荧光强度明显较弱，且GFP表达率分别为（21．5±2．7）％、（12．3±2．1）％和（0．2±0．1）％，均显著低于转染pFZD7-GFP的各对应组（P〈0．05）；对照组中，转染对照质粒pFZD7-Luc的肝癌细胞及L02细胞中未检测到GFP的表达，而转染对照质粒pEGFP-N1的各细胞株中均有大量细胞表达GFP。在裸鼠肝癌移植瘤中有相似的结果。结论FZD7启动子具有高度的肝癌特异性活性，T细胞因子／淋巴增强因子（TCF／LEF）转录因子结合位点在其转录调控中发挥关键作用。Objective To construct frizzled transmembrane receptor 7 （FZD7） gene promoter and study its tumor - specific transcriptional regulatory activity in hepatocellular carcinoma. Methods FZD7 promoter and the mutated FZD7 promoter （Mut FZD7 ） were constructed. Luciferase reporter vectors, pFZD7 - green fluorescent protein （GFP） and Mut pFZD7 - GFP were designed and synthesised. Hepato- cellular carcinoma ceils HepG2, SMMC7721 and normal liver ceils L02 were transfected with the recombi- nant vectors. The GFP expression of cells was detected through fluorescence microscopy and flow cytome- try. Nude mice models of human hepatocellular carcinoma xenograft tumor were established and injected with recombinant vectors. The green fluorescence was observed from tumor tissue sections through fluores- cence microscopy. Results The GFP was highly expressed in pFZD7 - GFP transfected HepG2 and SMMC7721 cells. The rates of HepG2 [ （45.1 ± 3.1 ） % ] and SMMC7721 [ （ 25.3 ± 2. 5 ） % ] cells ex- pressing GFP were significantly higher than that of L02 cells after pFZD7 - GFP transfection （ P 〈 0. 05 ）. The fluorescence intensity and expressing GFP rates in HepG2 [ （21.5 ± 2. 7）% ], SMMC7721 [ （12. 3 ± 2. 1 ） % ] and 1.02 [ （0. 2 ± 0. 1 ） % ] cells transfected with Mut pFZD7 - GFP was significantly lower than that of cells with pFZD7 - GFP transfection （P 〈0. 05）. However, GFP was not observed in hepatocellular carcinoma cell and L02 cells transfected with pFZD7 - Luc, while the resuhs in pEGFP - N1 groups were opposite. Similar results could also be obtained from tumor tissue sections after intratumoral injection of different vectors. Conclusion FZD7 promoter has a high degree of tumor - specific activity in hepatocellular carcinoma, and T - cell factor/lymphocyte enhancer factor （TCF/LEF） transcription factor binding sites play a key role in the transcriptional regulation.

关 键 词：肝细胞癌 卷曲蛋白跨膜受体7 启动子 肿瘤特异性 转录调控 

分 类 号：R394[医药卫生—医学遗传学]