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PRR11(proline-rich protein 11)是我们最近发现的一个新的肿瘤相关基因,在细胞周期和肿瘤发生等过程中起重要作用。该研究是在此前对PRR11启动子鉴定分析的基础上,对PRR11核心启动子区域中的核因子(nuclear factor Y,NF-Y)结合位点进行进一步的分析以确定其在PRR11转录调控中的作用。核苷酸序列同源性分析结果表明,PRR11核心启动子区域中的两个NF-Y结合位点在人、牛、大鼠和小鼠四个物种中均高度保守。共转染NF-Y表达质粒后,发现NF-Y的外源过表达可以明显提高PRR11的启动子活性。采用定点突变方法将PRR11启动子区域中的两个NF-Y结合位点单独或同时进行有效突变后,PRR11启动子活性明显下降,且NF-Y外源过表达对其启动子活性的激活效应也明显削弱甚至丧失。另外,对基因定点突变方法做出了改进,提出了一种更好的基于转录因子结合位点分析的碱基突变方法。这些结果表明,NF-Y结合位点是PRR11核心启动子区域中的重要的顺式作用元件,NF-Y可能通过调节PRR11的转录进而调节细胞周期和肿瘤发生等过程。
PRR11 (proline-rich protein 11) is a new tumor-associated gene that we recently discovered that plays an important role in cell cycle and tumorigenesis. Based on the previous analysis of the PRR11 promoter, this study further analyzed the nuclear factor Y (NF-Y) binding site in the PRR11 core promoter region to determine whether it is involved in PRR11 transcriptional regulation effect. Nucleotide sequence homology analysis indicated that the two NF-Y binding sites in the PRR11 core promoter region are highly conserved among four human, cow, rat and mouse species. After cotransfection of NF-Y expression plasmid, it was found that the exogenous overexpression of NF-Y can significantly improve the promoter activity of PRR11. Using site-directed mutagenesis to mutate the two NF-Y binding sites in the PRR11 promoter region individually or simultaneously, the PRR11 promoter activity was significantly decreased and the activation effect of the NF-Y exogenous promoter on its promoter activity Also significantly weakened or even lost. In addition, the method of site-directed mutagenesis has been improved, and a better base-mutation method based on the analysis of transcription factor binding sites has been proposed. These results indicate that the NF-Y binding site is an important cis-acting element in the PRR11 core promoter region. NF-Y may regulate the cell cycle and tumorigenesis by regulating the transcription of PRR11.