【作者】 刘春刚；

【导师】 李晓雪；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2010， 硕士

【摘要】 RASSF2是一个新发现的肿瘤抑制因子,是Ras相关信号通路中的负调控因子。很多证据表明,RASSF2与肿瘤的发生存在着密切的关联,在结肠癌、胃癌和鼻咽癌等癌变组织或肿瘤细胞中RASSF2是低表达的,但是在正常组织或细胞中RASSF2的表达却很高。有研究发现,RASSF2的低表达与DNA高甲基化相关。但是目前,有关RASSF2的抑癌分子机制仍所知甚少,而对于RASSF2基因表达调控的机制也了解不多。在本文中,我们发现组蛋白乙酰转移酶p300通过上调RASSF2的表达,抑制了胃癌细胞SGC-7901的增殖并对其可能的机制进行了深入的研究。我们的工作证实,过量表达p300能抑制胃癌细胞的增殖,并诱导细胞发生晚期凋亡;而这一过程可通过利用small interfering RNA (siRNA)降低RASSF2蛋白的表达水平而被逆转。染色质免疫沉淀(ChIP)实验证实,p300固有的乙酰转移酶活性在p300参与RASSF2的表达调控中是必需的;p300通过增强RASSF2启动子区域的组蛋白H3和H4的乙酰化水平促进RASSF2基因的启动子活性。而且p300和转录因子Sp1对RASSF2基因在启动子水平,mRNA水平及蛋白水平的表达具有协同激活的效果。针对Sp1和p300设计的siRNA能够通过部分地抑制Sp1和p300的表达而抑制RASSF2启动子的活性,并使RASSF2的mRNA和蛋白表达水平下降。ChIP的实验结果表明,转录因子Sp1能够促进p300被招募到RASSF2的启动子上,而过表达p300能够导致更多的转录因子Sp1与RASSF2启动子结合。我们推测,这可能与p300通过提高启动子处组蛋白H3和H4的乙酰化水平,使染色质结构变得更为松散有关。我们的研究工作将有助于更好的阐述RASSF2基因转录调控及其抑癌的分子机制,并为基于RASSF2的基因治疗提供一定的理论基础。更多还原

【Abstract】 RASSF2, a novel tumor-suppressor gene, is a member of Ras-associated protein family that negatively regulates Ras signaling. There has been increasing evidence that RASSF2 gene is related to tumorgenesis. In colorectal cancer (CRC) cells, gastric cancer cells, and nasopharyngeal cancer cells, the level of RASSF2 expression is lower, compared to normal tissues or cells. But little is known of the mechanism of RASSF2 gene regulation and its effect on inhibiting cell proliferation. In this study, we demonstrated that p300 was able to arrest cell proliferation and induce late apoptosis in gastric cancer cells SGC-7901; and this process was reversed by RASSF2 silencing through small interference RNA (siRNA). We showed that p300 participated in activation of RASSF2 expression in 293T and SGC-7901 cells. The chromatin immunoprecipitation (ChIP) assays verified that the intrinsic histone acetyltransferase (HAT) activity of p300 was essential for RASSF2 promoter activation through inducing the hyperacetylation of histone H3 and H4 at the RASSF2 promoter. Moreover, p300 cooperated with Sp1 to stimulate RASSF2 promoter activity, and increase its mRNA and protein expression. The siRNA-induced partial silencing of Sp1 and p300 resulted in a significant inhibition of RASSF2 promoter activity. The ChIP assays revealed that the ectopic expression of p300 promoted Sp1 binding to P2 and P3 and P4 regions of RASSF2A promoter. Meanwhile, the recruitment of p300 on the P2 and P3 regions was also enhanced significantly after Sp1 overexpression. These data will contribute to elucidating the mechanisms of RASSF2 transcriptional control and its ability of suppressing cancer cells proliferation, which is crucial to the development of new therapeutic strategies for RASSF2-related gene therapy.更多还原