摘要
目的 探讨人子宫内膜癌细胞株HEC 1A细胞ras基因第 12密码子中突变型 (即[12 Asp]K ras4B基因 )对雌激素受体 (ER)及其亚型α、β蛋白的表达和转录活性的影响。 方法 ( 1)用蛋白免疫印迹法检测 [12 Asp]K ras4B基因对ERα、ERβ蛋白表达的影响。 ( 2 )构建含荧光素酶报告基因和ER反应元件 (ERE)的真核表达质粒 pGL3 ERE luciferase ,与表达绿色荧光蛋白的 pEGFP N1质粒共同转染鼠成纤维细胞株NIH3T3细胞及HEC 1A细胞 ,观察雌二醇调节 [12 Asp]K ras4B基因对ER的转录活性 ;分别转染含有全长野生型ERαcDNA的 pSV5 HER0和抑制raf基因的 pCMV rafS6 2 1A ,探讨 [12 Asp]K ras4B/raf信号通路对ER转录活性的调节作用。 结果 ( 1)NIH3T3细胞转染 pcDI [12 Asp]K ras4B质粒与转染空载体 pcDI质粒比较 ,ERα表达水平增加 3 6倍 (分别为 97± 2 5和 349± 6 7,P <0 0 1) ,ERβ增加 1 9倍 (分别为 12 8± 37和 2 4 9± 30 ,P <0 0 5 )。 ( 2 ) pCMV rafS6 2 1A质粒分别转染到含 pcDI [12 Asp]K ras4B质粒的NIH3T3细胞和HEC 1A细胞 ,转染前、后NIH3T3细胞ERα蛋白表达水平分别为 72 4± 4 5和 310± 4 6 (P <0 0 5 ) ,ERβ蛋白表达水平分别为 4 93± 2 0和2 84± 2 0 (P <0 0 1) ;转染前、后HEC
Objective To investigate the effect of mutant type [ 12 Asp]K ras4B gene on the expression of estrogen receptor(ER) α and β and their transcriptional activity as a transcription factor in endometrial carcinoma HEC 1A cell line Methods (1) Effect of [ 12 Asp]K ras4B on the expression of ER α and β were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3 luciferase ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co transfected into NIH3T3 and HEC 1A cell lines with pEGFP N1 to examine the effect of [ 12 Asp]K ras4B on ER transcription that is regulated by estradiol In addition, they were transfected into pSV5 HER0 (containing full length wide type ERα cDNA) and pCMV rafS621A (inhibiting raf kinase) plasmids to test the effect of [ 12 Asp]K ras4B/raf signal pathway on transcriptional activity of ER proteins Results (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3 6 fold (97±25, 349±67, P <0 01) and 1 9 fold (128±37, 349±30, P <0 05) in ERα and ERβ, respectively, in pcDI [ 12 Asp]K ras4B NIH3T3 cells after transfection. (2) In pcDI [ 12 Asp]K ras4B NIH3T3 cells, the ratios for ERα and and ERβ levels before transfection of rafS621A plasmids to that after the trasfection, were 2 4∶1 (724±45, 310±46, P <0 05) and 1 8∶1 (493±20, 284±20, P <0 01), respectively; In HEC 1A cells, these ratios were 2 1∶1 (566±22, 279±30, P <0 01) and 2 4∶1 (405±33, 165±15, P <0 01), respectively. (3) In low serum (2%) culture condition, estradiol (E 2) stimulated luciferase activity with an increase of 13 fold (130±42, 1681±242, P <0 01) in pcDI [ 12 Asp] K ras4B NIH3T3 cells, 19 fold (141±39, 2644±331, P <0 001) in HEC 1A cells, respectively, when compared with those in the absence of E 2. (4) In pSV5 HER0 transfected pcDI [ 12 Asp] K ras4B NIH3T3 cells and HEC 1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly The luciferase activity was increased for 8 fold (1048±91, 8099±452, P <0 01) and 6 fold (2148±259, 12 705±2670, P <0 001), respectively. rafS621A mutant had suppressive effects on luciferase activities in HEC 1A cells and pcDI [ 12 Asp]K ras4B NIH3T3 cells The ratio of luciferase activities in pcDI [ 12 Asp]K ras4B NIH3T3 and HEC 1A cells, before and after transfection was 7 8∶1 (1184±168, 152±27, P <0 05) and 6 4∶1 (1949±212, 304±60, P <0 01), respectively Conclusions (1) [ 12 Asp]K ras4B can enhance the expression of ERα and β proteins This may be correlated with [ 12 Asp]K ras4B/raf signaling pathway. (2) The effect of mutant type [ 12 Asp]K ras4B gene on ERs transcriptional activity in HEC 1A cells appears to need E 2
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2004年第1期30-34,共5页
Chinese Journal of Obstetrics and Gynecology
基金
首都医学发展基金资助项目 (ZD199911)
国家自然科学基金资助项目(3 0 0 0 0 178)
关键词
子宫内膜肿瘤
肿瘤细胞
培养的
受体
雌激素
遗传转录
遗传转录
Endometrial neoplasms
Tumor cells, cultured
Receptors, estrogen
Transcription, genetic
Genes, ras
