胰岛素样生长因子结合蛋白3增强子元件(IEE)的生物学特征

Biological characteristics of a enhancer element of insulin-like growth factor binding protein-3 (IEE)

背景:在细胞水平,胰岛素样生长因子结合蛋白3竞争性地与胰岛素样生长因子结合阻止了胰岛素样生长因子与其受体结合,从而抑制了胰岛素样生长因子的活性。目的:观察衰老细胞中调节性蛋白质胰岛素样生长因子结合蛋白3的生物学特征。设计、时间及地点:单一样本观察,于2006-09/2007-09在西安交通大学生物医学信息工程教育部重点实验室完成。材料:人胚肺二倍体成纤维细胞(2BS)购于中国生物制品研究所。方法:用Northern的方法显示胰岛素样生长因子结合蛋白3基因的表达在年轻和衰老的2BS细胞中存在差异;聚合酶链反应扩增出人类胰岛素样生长因子结合蛋白3上游包括5'-UTR区的2kb的序列,并用酶切得到4组不同长短的胰岛素样生长因子结合蛋白3启动子片段并确定可调控转录活性的区域;通过重叠寡核苷酸凝胶阻滞实验确定在该活性区域中的增强子元件-IEE(IGFBP-3enhancer element)及其与蛋白结合的碱基序列等;用DNase I Footprinting法确定了IEE中与蛋白结合的核心序列。主要观察指标:①胰岛素样生长因子结合蛋白3基因在年轻和衰老细胞中的表达差异。②通过重叠寡核苷酸确定增强子元件。③通过凝胶阻滞试验证实该复合物结合活性与衰老相关的。④通过凝胶阻滞试验确定IEE与蛋白结合的碱基序列。⑤用DNase I Footprinting法确定IEE中与蛋白结合的核心序列。⑥判断与IEE结合蛋白的相对分子质量。结果:与年轻的2BS细胞相比,衰老的2BS细胞中胰岛素样生长因子结合蛋白3基因的表达升高;5'-cca gcc tgc caa gca gcg tgc ccc ggt tgc-3'是胰岛素样生长因子结合蛋白3的增强子元件;该元件与蛋白结合的核心序列是CTG CCA和GCG TGC CCC G,而且这种结合是与衰老相关的;结合蛋白的相对分子质量大约在27000。结论:在2BS细胞中发现了一个新的转录增强子,它与蛋白结合的核心序列是CTG CCA和GCG TGC CCC G,可与一个大约27000的蛋白结合;在衰老细胞中可选择性地促进胰岛素样生长因子结合蛋白3的表达。

BACKGROUND： At the cellular level, insulin-like growth factor binding protein-3 （IGFBP-3） inhibit the activity of insulin-like growth factors （IGFs） via competitively binding IGFs and thereby preventing their binding to the IGF receptor. OBJECTIVE： To study the Biological characteristics of IGFBP-3 in senescent cells. DESIGN, TIME AND SETTING： A single sample observation was performed at the Key Laboratory of Biomedical Information Engineering, Ministry of Education, Xi＇an Jiaotong University between September 2006 and September 2007. MATERIALS： Human embryonic lung diploid fibroblasts （2BS） were obtained from the National Institute of Biological Products, Beijing, China. METHODS： The difference of IGFBP-3 expression in young and senescent 2BS cells were displayed by Northern blot analysis. The 2 kb human IGFBP-3 promoter containing 5＇-UTR was generated by PCR amplification and digested into four fragments by the restriction endonuclease. Further mobility shift analysis showed that the lEE reduced or abolished complex formation and markedly reduced IGFBP-3 promoter activity in senescent cells. Then DNase I Footprint Identified the protein-binding core sequence within the lEE. MAIN OUTCOME MEASURES： ①The differential expression of IGFBP-3 in young and senescent 2BS cells. ② Enhancer element was identified by overlapping oligonucleotides. ③The correlation of the protein binding activity and senescence-dependent was investigated by gel retardation experiments. ④The base sequence within the lEE responsible for protein binding was identified with gel retardation experiments. ⑤The protein-binding core sequence within the lEE was identified with DNase I Footprint. ⑥The molecular weight of the protein binding lEE. RESULTS： IGFBP-3 expression increased substantially in senescent cells compared with young cells; 5＇-ccagcctgccaagcagcgtgccccggttgc-3＇ was the enhancer element of IGFBP-3; the protein-binding core sequence within the lEE was CTGCCA and GCGTGCCCCG, which showed a senescence-dependent manner; the molecular mass of the protein was expected to be 27000. CONCLUSION： lEE is a new enhancer element of IGFBP-3 found in 2BS, core sequence of which is CTGCCA and GCGTGCCCCG combining with molecular mass of 27 000 protein. The newly found lEE can increase the IGFBP-3 gene expression in senescent 2BS cells.