BRD7调控Rb/E2F通路的分子机制研究

The Molecular Mechanisms of BRD7 Gene in Regulating of Rb/E2F Pathway

BRD7是采用cDNA代表性差异分析法克隆的一个新的Bromodomain基因,过表达BRD7可抑制鼻咽癌细胞的生长和细胞周期进程,同时发现BRD7基因可以调控Rb/E2F通路的活性.该研究旨在进一步探讨BRD7调控Rb/E2F通路的分子机制.通过蛋白质印迹和RT-PCR实验方法发现,BRD7能够降低Rb的磷酸化水平,抑制cyclinD1、cyclinE的蛋白质表达,上调CDK4抑制子P19的mRNA表达,但对CDK4和CDK2的蛋白质表达没有明显影响;通过荧光素酶实验从转录调控水平进一步证实了BRD7能够明显抑制cyclinD1启动子活性;采用反义核酸技术抑制COS7细胞内源性BRD7的表达后,发现cyclinD1、cyclinE、磷酸化Rb的蛋白质表达水平上调,并且可以促进细胞生长.这些结果表明:BRD7参与调控Rb/E2F信号通路中重要靶分子的表达,抑制Rb/E2F通路的活性,从而阻止细胞周期G1-S期进程,抑制鼻咽癌细胞生长.

BRD7 is a novel bromodomain gene isolated by cDNA representational difference analysis （GenBank accession number： AF 152604）. Ectopic expression of BRD7 inhibited NPC cell growth and cell cycle progression. Previous studies demonstrated that BRD7 gene could regulate the activity of Rb/E2F pathway. In order to further explore the molecular mechanisms of BRD7 regulating Rb/E2F pathway, Westernblot and RT-PCR analysis were carried out. Results showed that BRD7 could inhibit the phosphorylation of Rb, decrease the expression of cyclin D1 and cyclin E, up-regulate p19 in RNA level, but had no effects on the expression of CDK4 and CDK2. Luciferase reporter assay suggested that BRD7 repressed cyclin D1 promoter activity. Furthermore, an antisense nucleic acids technology was performed to silence the endogenous BRD7 gene in COS7 which resulted in the upregulation of cyclin D1, cyclin E, phosphorylated Rb, and acceleration of the cell growth. As a result of this research, BRD7 inhibits G1-S phase progression in cell cycle via regulating the important molecules involved in Rb/E2F pathway.