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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 1A细胞ER
Objective To investigate the effect of mutant (12 Asp) K ras4B gene on the estrogen receptor (ER) and its subtypes α and β in ras gene of human endometrial carcinoma cell line HEC 1A Effects of transcriptional activity. Methods (1) The effect of [12 Asp] K ras4B gene on the expression of ERα and ERβ protein was detected by Western blotting. (2) The eukaryotic expression plasmid pGL3 ERE luciferase containing luciferase reporter gene and ER reaction element (ERE) was constructed and transfected into NIH3T3 cells and HEC 1A cells with pEGFP N1 plasmid expressing green fluorescent protein. Estradiol was used to modulate the transcriptional activity of [12 Asp] K ras4B gene in ER. Transfection of pSV5 HER0 containing full-length wild-type ERα cDNA and pCMV rafS6 2 1A containing raf gene were carried out to investigate the [12 Asp] K ras4B / raf signal Pathway Regulates ER Transcriptional Activity. Results (1) Compared with pcDI plasmid transfected with pcDI [12 Asp] K ras4B plasmid, NIH3T3 cells transfected NIH3T3 cells increased the expression of ERα by 36% (97 ± 2 5 and 349 ± 6 7, P 0 01, respectively ), ERβ increased by 19 times (12 8 ± 37 and 249 ± 30 respectively, P <0 05). (2) The pCMV rafS6 2 1A plasmids were transfected into NIH3T3 cells and HEC 1A cells containing pcDI [12 Asp] K ras4B plasmids respectively. The expression levels of ERα in NIH3T3 cells before and after transfection were 72 4 ± 45 and 310 ± 4β (P <0 05). The expression of ERβ protein was 433 ± 20 and 2 84 ± 20 (P <0.01) respectively. Before and after transfection, the expression of ERβ