论文部分内容阅读
在TPA诱导永生化食管上皮细胞癌变中,NGAL(neutrophil gelatinase-associated lipocalin)过表达,提示食管癌细胞NGAL转录调控区可能存在TPA应答元件.为了对食管癌细胞NGAL的这一TPA应答元件进行分段定位鉴定,首先将NGAL5′侧翼区不同长度片段-1 431~+84-、1 137~+84、-945~+84、-657~+84、-416~+84和-152~+84等依次插入质粒pGLP,构建NGAL/pGLP系列报告基因荧光素酶表达载体pGLP-1431、pGLP-1137、pGLP-945、pGLP-657、pGLP-416和pGLP-152;然后将上述质粒分别同pRL-TK共转染食管癌细胞EC109,最后用5 ng/ml的TPA刺激转染的EC109,检测报告基因荧光素酶活力,综合判定报告基因荧光素酶活力变化趋势,并以此对食管癌细胞NGAL5′侧翼区TPA应答元件进行分段定位.实验结果表明,食管癌细胞NGAL5′侧翼-657~-417区段内存在着较强的TPA应答元件.生物信息学分析显示,NGAL5′侧翼-657~-417区段至少存在3个潜在的TPA应答元件,表明有应答TPA刺激的结构基础.这些研究有助于从分子水平揭示TPA诱导永生化食管上皮细胞癌变中NGAL基因过表达机制.
Overexpression of neutrophil gelatinase-associated lipocalin (NGAL) in TPA-induced immortalized esophageal epithelial cells suggests that TPA-responsive elements may exist in the NGAL transcriptional regulatory region of esophageal cancer cells.To screen this TPA response element of esophageal cancer cells, NGAL, First of all, the fragments of different lengths of NGAL 5 ’flanking region, such as -1 431 ~ + 84-, 1 137 ~ + 84, -945 ~ + 84, -657 ~ + 84, -416 ~ + 84 and -152 ~ + 84 Then inserted into the plasmid pGLP to construct the luciferase expression vector pGLP-1431, pGLP-1137, pGLP-945, pGLP-657, pGLP-416 and pGLP-152 of NGAL / pGLP series reporter gene; TK was co-transfected into EC109 esophageal cancer cells and the transfected EC109 was stimulated with TPA at a concentration of 5 ng / ml. The reporter gene luciferase activity was detected and the luciferase activity trend of the reporter gene was determined synthetically. TPA response element in the flanking region.The experimental results showed that there was a strong TPA response element in the -657 ~ -417 segment of the esophageal cancer cell line NGAL5’.Bioinformatics analysis showed that -657 ~ There are at least 3 potential TPA response elements in the -417 segment, indicating there is A structural basis of TPA stimulated. These studies help reveal immortalized esophageal epithelial carcinogenesis mechanism of NGAL over the TPA-induced gene expression at the molecular level.