摘要
探讨过表达特异AT序列结合蛋白-1(special AT-rich sequence binding protein,SATB1)核基质结合区(MAR)结合蛋白对胰岛素样生长因子结合蛋白-2(IGFBP2)基因表达的影响,并对其影响机制进行初步探索.首先用脂质体将SATB1的真核表达载体pcDNA3.1-SATB1转染至K562细胞,通过6周G418的筛选获得阳性克隆,RT-PCR、实时PCR及Western印迹验证过表达情况,对阳性克隆细胞中IGFBP2的表达用RT-PCR、实时PCR及Western印迹方法进行检测;然后用RNAi的方法干扰阳性细胞中SATB1的表达后,同样用上述3种方法再次检测IGFBP2的表达状况;用生物信息学方法对IGFBP2基因进行MAR序列与SATB1结合位点搜索分析,寻找SATB1影响IGFBP2基因表达的机制.结果显示,在稳定转染的情况下,实验组K562-SATB1细胞与转染空载体pcDNA3.1的K562-3.1细胞和未转染细胞K562相比,IGFBP2 mRNA水平上调了近7倍,而蛋白水平变化不明显.RNA干扰后,IGFBP2的表达在mRNA水平也相应下调,蛋白水平的变化同样不明显.通过生物信息学分析发现,IGFBP2第1个内含子中可能存在2.5 kb MAR样序列,且MAR样序列上存在多个SATB1的潜在结合位点.综上所述,过表达SATB1可以使K562细胞中IGFBP2 mRNA表达水平提高,而且其调控机制可能与SATB1直接和IGFBP2基因中的MAR样序列结合有关.
To detect the effect of over-expressing special AT-rich sequence binding protein （SATB1） which is a matrix attachment region （MAR） binding protein on insulin-like growth factor binding protein 2 （IGFBP2） expression and explore the possible mechanisms, the SATB1 expression plasmid pcDNA3.1-SATB1 and the empty vector pcDNA3.1 were separately transfected into human erythroleukemia K562 cells by lipofeetamine 2000. Through G418 selection after 6 weeks we got the positive clones, and confirmed by RT-PCR, real-time PCR and Western blotting, then examined the expression of IGFBP2 under stable expression of SATB1 by RT- PCR, real-time PCR and Western blotting. In order to research the relations between SATB1 and IGFBP2,RNA interference experiments were performed, then the expression of IGFBP2 was detected by the same methods. After that, we applied the bioinformatics software to analyze the whole sequence of IGFBP2. The results showed that the SATB1 could enhance the IGFBP2 gene expression nearly 7 folds on mRNA level in K562 cells, and httle effect on protein level, and silenced expression of SATB1 also reduced the IGFBP2 gene expression in mRNA level. After the analyses by bioinformatics, we found that there were a 2.5 kb MAR-like sequence in the first intron of IGFBP2, and there were several putative SATB1 binding sites in the MAR-like sequence. The results showed that over-expressing SATB1 could enhance the IGFBP2 gene expression in mRNA level in K562 cell, and the possible regulation mechanism may be through the direct effect of SATB1 and the MAR-like sequence within IGFBP2 gene .
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2009年第2期169-176,共8页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金(No.3030066)
国家重点基础研究发展规划(973计划
No.2004CB518804)
上海市科委项目(No.04JC14041andNo.07JC14043)
仁济医院基础和应用研究合作基金(No.PY07001)资助~~
