DNMT1基因对cAMP诱导恶性胶质瘤定向分化的作用及其机制

cAMP能诱导恶性胶质瘤分化，但分子机制仍不清楚。我们预实验发现，cAMP可负性调控胶质瘤中DNMT1的mRNA和蛋白的稳定性，并能同时诱导星形胶质细胞分化标志GFAP及神经元分化标志MAP2、TUBB3的表达。进一步发现，过表达DNMT1能阻断cAMP诱导的MAP2的表达，增强GFAP的表达。机制研究发现，cAMP能显著增强神经元特异性转录因子Ascl1/Brn2/Ngn2的表达，且Ascl1/Brn2/Ngn2基因启动子富含CpG岛。由此我们提出假说：cAMP在转录后和翻译后两个层面负性调控DNMT1蛋白水平，解除甲基化对Ascl1/Brn2/Ngn2启动子的转录抑制作用，诱导胶质瘤向神经元方向分化。本研究拟通过CRISPR、定点突变、重亚硫酸盐测序及两种在体肿瘤模型阐明DNMT1调控胶质瘤定向分化的分子机制，最终为研发以DNMT1信号通路为靶点的新型胶质瘤定向诱导分化剂提供理论依据。

cAMP was demonstrated to induce differentiation of malignant glioma, however, the underlying mechanisms are still yet to be clarified. Our pilot experiment indicates that cAMP can negatively regulate the stability of DNMT1 mRNA and protein. It can efficiently induce expression of both GFAP which is the biomarker of astrocyte as well as MAP2 and TUBB3 which are the well-known biomarkers of neuron. Further investigation shows that overexpression of DNMT1 not only blocks the cAMP induced MAP2 expression, but also enhances the cAMP induced GFAP expression. Mechanistically, cAMP can strongly stimulate expression of three neuron- specific transcriptional factors Ascl1/Brn2/Ngn2 which contain several CpG islands resided on their promoters. Thus, we propose a hypothesis that cAMP stimulators can negatively regulate DNMT1 protein and mRNA at both post-transcriptional and post-translational levels, which in turn releases the methylation-mediated Ascl1/Brn2/Ngn2 silence and induces differentiation of malignant glioma into neuron. The current project is to use CRISPR, Side-Directed Mutagenesis , Bisulfite Genomic Sequence Assay and two different animal models to fully clarify the underlying mechanism of DNMT1 regulated glioma differentiation. Our study will eventually provide a new theoretical support for the development of a novel glioma differentiation inducing agent that targets DNMT1 signal pathway.