摘要
临床流行病学研究显示,ERα基因第5外显子缺失突变体ERδ5在原发性肝细胞癌特异性高表达,它被认为是判断肝癌预后的最重要的新型分子标志.本研究从肝癌细胞分离雌激素受体突变体ERδ5基因,并对其进行克隆、表达,探讨其分子生物学特性及生物学功能.通过反转录PCR,从肝细胞分离出ERδ5基因并将其克隆到真核表达载体,通过体外翻译及Western印迹验证该基因成功表达;通过细胞荧光技术检测了ERδ5在肝细胞中的定位;利用荧光素酶报告基因检测技术确证ERδ5对ERα转录活性的影响.结果发现,从肝癌细胞分离出长1113bp的ERδ5基因,该基因编码蛋白在体外不稳定,易于降解.在肝癌细胞中,ERδ5蛋白主要定位在细胞核,与ERα定位极为相似,但在生物学功能上,ERδ5能够明显干扰ERα的转录活性.研究结果表明,ERδ5作为显性阴性突变体,在肝癌细胞中直接抑制ERα的转录活性.
Clinical epidemiology data have shown that the ERα mutant form with the entire exon 5 deleted （ERAS） is preferentially expressed in patients with HCC compared with patients with normal livers, which is the strongest negative predictor of survival in operable HCC. ERδ5gene was isolated from liver tumor cells by RT-PCR. ERδ5protein expression was detected by in vitro translation and Western blotting. ERδ5 subcellular location was visualized in hepatocytes by fluorescence analysis. The effect of ERδ5on ER transcription was investigated by luciferase reporter assay. ERδ5 gene of 1 113 bp length was successfully isolated from SMMC7721 ceils by RT-PCR. However, ERδ5 protein was very unstable in vitro. ERδ5 protein was mainly located in nucleus of SMMC7721 cells. ERδ5 could functionally inhibit the transcriptional activity of ERα in SMMC7721 cells. These results suggested that ERδ5, a dominant-negative variant of ERα, could significantly inhibit the transcriptional activity of ERα, in SMMC7721 cells.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2006年第8期635-639,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家863计划(No.2002BA71102-5)
国家自然科学基金[No.30571655
30530320]
中国博士后基金第35批资助项目~~
关键词
肝细胞癌
雌激素受体
变异
分子克隆
转录活性
hepatocellular carcinoma
estrogen receptor
mutant
molecular cloning
transcriptional activity
