Atrx 基因失活促进脑胶质瘤形成的表观遗传学机制研究

Given that one key event in glioma pathogenesis is the corruption of cell differentiation programs, it stands to reason that glioma cells may depend on distinct epigenetic pathways to sustain aberrant self-renewal against differentiation, and therefore be uniquely sensitive to certain perturbations of epigenetic structures. So using the epigenetic mechanisms to intervene and restore to their normal state by epigenetic therapy, makes such initiatives promising and therapeutically relevant. Previously, we used a customized shRNA library targeting 243 known chromatin regulators and performed the primary screen using one-by-one approach in three independent derived mouse glioma tumor-initiating lines. During the screening, we found that knockdown of Atrx by shRNAs promotes glioma initiating cells self-renewal and surpress the differentiation, indicating that Atrx is a potential glioma tumor suppressor. In this project, we are going to use Atrx shRNA to downregulate the Atrx expression in mouse glioma tumor-initiating cells and mouse neural stem cells, Analysis the effects of the differentiation and proliferation under the differentiation condition. Then we will perform RNA-seq and CHIP-sequencing to understand the epigenetics molecular mechanism of Atrx during glioma pathogenesis. This project will provide theoretical basis and experimental basis for the the invention of novel therapeutics informed by an enhanced understanding of its molecular pathogenesis.

胶质瘤细胞依赖表观遗传途径维持异常更新，抑制细胞正常分化，成为恶性胶质瘤发病机制关键环节之一。利用表观遗传学机制干预，恢复肿瘤细胞表观遗传修饰正常状态，可为恶性胶质瘤治疗开辟新途径。在前期工作中，我们利用三种鼠源性胶质瘤始动细胞（GIC）对靶向243种染色质调控子shRNA表观遗传学文库逐一筛查，发现下调Atrx基因表达，能够明显促进小鼠GIC增殖，抑制其分化，故推测Atrx是潜在脑胶质瘤抑癌基因。在本项目中，我们拟利用RNA干扰技术下调Atrx基因表达，观察在分化条件下，其对小鼠GIC及神经干细胞(NSC)的增殖、分化以及脑胶质瘤发生产生的影响。并以此为基础，利用细胞免疫染色，ChIP 测序及RNA测序等技术，从分子、细胞及动物模型水平，揭示Atrx基因缺失促进脑胶质瘤形成的表观遗传学分子机制。为深化对脑胶质瘤发病机理的认识，推进针对脑胶质瘤表观遗传学新疗法的开发提供理论基础与实验数据。

胶质瘤细胞依赖表观遗传途径维持异常更新，抑制细胞正常分化，成为恶性胶质瘤发病机制关键环节之一。利用表观遗传学机制干预，恢复肿瘤细胞表观遗传修饰正常状态，可为恶性胶质瘤治疗开辟新途径。我们利用三种不同小鼠脑胶质瘤始动细胞对shRNA表观遗传学文库进行初步筛选，发现下调Atrx基因可以抑制脑胶质瘤始动细胞分化，促进肿瘤始动细胞增殖。通过组织微阵列和免疫组化技术对216例多形性胶质母细胞瘤进行ATRX蛋白的检测，发现36% 的病例中ATRX蛋白发生缺失。Atrx shRNA 转染小鼠神经干细胞后，Brdu掺入实验显示Atrx下调促进神经干细胞细胞增殖，对照组神经干细胞很快分化，失去Nestin表达，而Atrx下调的细胞则保持Nestin染色阳性，故推测Atrx可能是潜在的脑胶质瘤抑癌基因。在分化条件下，下调Atrx可以明显促进小鼠胶质瘤始动细胞的生长，同时抑制神经干细胞分化。体内实验证实Atrx基因下调对脑胶质瘤始动细胞可以促进增殖，抑制分化。同时Atrx表达沿成人神经元谱系分化途径逐渐升高的模式表明Atrx在诱导和/或维持神经元分化状态中起积极作用。尽管Atrx对神经元谱系发展有影响，但下调似乎不影响分化后的神经元谱系的组成。我们还发现Atrx缺失能够诱导小鼠IDH1R132H转染的p53-/-NPCs在将其原位移植到受体小鼠后的神经胶质瘤的转化。为了解Atrx在神经元分化中所起的作用，通过RNAseq检测了分化的IDH1R132H转染的p53-/-NPC的基因表达谱。进一步分析显示这些分化相关基因，相对于它们的对照，Atrx突变的NPC的分化过程中分化相关基因表现出明显的减弱或抑制。这些数据进一步支持了Atrx促进NPC分化的功能。ATRX是一种可能在神经发育调节中起作用的神经胶质瘤抑制因子，ATRX神经元分化所必需的，它的下调促进脑胶质瘤的成瘤能力。为深化对脑胶质瘤发病机理的认识，推进针对脑胶质瘤表观遗传学新疗法的开发提供理论基础与实验数据。

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