SATB1-SOX9调控葡萄膜黑色素瘤增殖分化的表观遗传机制

Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults; however, the overall death rate for UM remains high because of the development of metastatic disease, which is highly resistant to chemotherapy. One reason for broad treatment resistance is that most melanoma drugs have cell cycle exit by apoptosis as a common intended action, however, master regulators of such cell cycle exits, e.g., p53, p16, are frequently genetically deleted from UM cells. To address this common mechanism of resistance, there is a need to identify melanocyte proliferation-terminating genes and pathways which are epigenetically instead of genetically suppressed: such genes/pathways would at least in theory be available for activation, especially if the mechanisms by which they come to be aberrantly epigenetically repressed are elucidated. Previous studies demonstrated that SATB1-SOX9 pathway plays an important role in regulating proliferation and differentiation of cutaneous melanoma (CM). UM and CM interrogation in parallel could be mutually informative in identifying pathways and events which are essential for melanocyte maturation and transformation. In contrast, there is paucity of data regarding such pathway in UM. In this research, the melanocyte maturation driver SOX9 was confirmed highly expressed in a TET-ON system, we intend to investigate the underline molecular mechanisms involved in maturation and differentiation in TET-ON-SOX9 UM cells by epigenetic means. Confirmation the regulation and elucidation of the SATB1-SOX9 pathways by which it is produced could suggest rational strategies to restore proliferation and differentiation in UM. This study is further expected to predict UM survival by SATB1 expression, and to offer an alternative to conventional therapies that attempt to activate apoptosis genes that may be genetically unavailable.

葡萄膜黑色素瘤（uveal melanoma, UM）是成人中最常见的恶性眼内原发性肿瘤，对化疗药耐药性高。这些药物大多作用于细胞周期依赖的细胞凋亡机制，然而调节细胞周期的关键基因，例如：p53、p16在多数UM肿瘤中突变或者缺失。为了寻找UM耐药的根本原因，有必要寻找与黑色素细胞增殖分化相关、非凋亡依赖的基因/通路。综合文献报道，SATB1-SOX9信号转导通路能调控皮肤黑色素瘤（Cutaneous melanoma, CM）的增殖分化，但对于眼部UM生长的表观遗传调控机制，目前尚不清晰。本研究通过建立黑色素晚期分化基因SOX9高表达的UM细胞模型，诱导肿瘤细胞分化成熟的表观遗传学现象。揭示SATB1对SOX9的调控关系以及SATB1-SOX9信号转导通路对于UM增殖分化的表观遗传学调控机制，并进一步探讨SATB1表达水平与UM患者生存时间的关系，为传统诱导肿瘤凋亡的抗瘤模式提供新思路。

近年来，尽管对葡萄膜黑色素瘤（uveal melanoma, UM）的治疗手段层出不穷，但是UM的死亡率并未随之减少。其中，治疗抵抗重要的原因之一是由于大多治疗UM的药物作用于细胞周期依赖的细胞凋亡机制。然而，在遗传学上，调节细胞周期的关键基因，例如：p16/CDKN2A在多数UM肿瘤中突变或者缺失。为了寻找UM耐药的根本原因，有必要寻找与黑色素细胞增殖分化相关、非凋亡依赖的基因/通路，在UM的表观遗传学上而不是遗传学上被抑制。此种被抑制的基因/通路理论上至少能被可逆激活，并且阐明存在此种表观遗传学上被抑制的状态的机制显得尤为重要。. 肿瘤基因MYC促进黑色素细胞的增殖。通常，MYC与黑色素细胞发育过程中表达增高的蛋白相互拮抗，使这些拮抗蛋白的表达受抑制。因此，葡萄膜黑色素瘤中这些MYC-拮抗蛋白被抑制的现象可能预示UM的致病机理。本研究采用TET-ON系统，通过诱导剂Doxycycline激活UM细胞中低表达的SOX9基因，以拮抗MYC介导的恶性肿瘤细胞增殖。我们观察到UM细胞株MUM2B转染SOX9后，能快速有效地抑制MYC的功能表达。同时，我们探索了一种引起这种表观遗传学抑制可能的原因：SATB1基因的调控。通过验证SATB1特异绑定SOX9增强子和启动子DNA调控元件区域的一系列研究，我们着重进行SATB1基因功能异常是否与黑色素细胞分化基因表观遗传学抑制相关的一系列研究，包括SATB1基因突变的检测、SATB1突变与UM CPG甲基化关系、SATB1表达水平与患者生存时间的多变量分析以及体内实验检测SOX9对肿瘤发生的影响。. 本研究通过建立黑色素晚期分化基因SOX9高表达的UM细胞模型，探讨上调UM细胞SOX9基因的表达，诱导肿瘤细胞分化成熟的表观遗传学现象。揭示SATB1对SOX9的调控关系以及SATB1-SOX9信号转导通路对于UM增殖分化的表观遗传学调控机制，并进一步 探讨SATB1表达水平与UM患者生存时间的关系。为取代试图激活凋亡，却事实上不可行的传统抗瘤方法，提供临床肿瘤治疗科学理论依据，为传统诱导肿瘤凋亡的抗瘤模式提供新思路。