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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 epidemiological studies have shown that ERδ5, a deletion mutant of exon 5 of ERα gene, is highly expressed in primary hepatocellular carcinoma and is considered as the most important new molecular marker for prognosis of hepatocellular carcinoma.In this study, The ERδ5 gene was isolated from estrogen receptor mutant and cloned and expressed, and its molecular biological characteristics and biological functions were explored.The ERδ5 gene was isolated from hepatocytes by RT-PCR and cloned into eukaryotic expression The expression of ERδ5 in hepatocytes was detected by cytofluorometry and the effect of ERδ5 on the transcriptional activity of ERα was confirmed by luciferase reporter gene assay.It was found that from the liver cancer The gene of ERδ5 with a length of 1113bp was isolated and its protein was unstable and easily degradated in vitro.In the hepatocellular carcinoma cells, the ERδ5 protein mainly localized in the nucleus and was very similar to the ERα localization, but ERδ5 could significantly interfere with the biological function ERα transcription activity.The results show that ERδ5 as a dominant negative mutant in liver cancer cells directly inhibit ERα transcriptional activity.