物理机械力信号上调肿瘤干细胞Sox-2表达的表观遗传修饰研究

The bottom-neck in tumor stem cell research resides in the limitations of the existing cell isolation methods. The stem cells that isolated by these methods are usually insufficient to match the requirement of both the quantity and the purity. In the previous study, we used 3D soft matrix for cell culture, based on which mechanical force was generated, and efficiently isolated tumor stem cells. However, the innate mechanisms of substrated stiffness related mechanical force in regulating relf-renewal of tumor stem cells is still under estimation. As indicated by other groups, and our in house data, we predict here, that substrate stiffness change cause regulation of H3K9 methylation, and in turn effects tumor stem cells on either sustentation of self-renewal or induction of differentiation. This project is designed to elucidate the exact mechanism by which soft substrate sustains relf-renewal of tumor stem cells. We will thus focus on the study of tumor stem cells, and start from Sox-2 expression. This project will be carried out firstly, to explore the impacts of substrate stiffness on Sox-2 expression and tumor stem cell self-renewal sustentation, as well as the key roles of Sox-2 expression in self-renewal of tumor stem cells. And secondly, using ChIP and methylation related-enzyme inhibitors, to examine both the regulating roles and mechanisms of H3K9 methylation in Sox-2 expression and tumor stem cell self-renewal. At last, to further optimize and update the now-existing protocol for tumor stem cell cultures, through adjusting mechanical force signals, and reproof the H3K9 methylation-mediated regulating mechanisms of self-renewals in vivo. This study is featured by utilizing mechanical signals, and will provide novel theoretical guidelines for the emerging research fields of tumor stem cell-based epigenetic modifications, as well as novel therapeutic strategies.

肿瘤干细胞研究的瓶颈在于其分离纯化较难满足数量和纯度等要求。申请人前期采用软基质提供物理机械力信号，成功将肿瘤干细胞筛选和扩增，然而物理信号诱导其Sox-2表达与自我更新的机制尚不明确。基于文献报道和申请人研究结果，我们推测，基质的物理信号通过调控组蛋白H3K9甲基化，以影响肿瘤干细胞维持干性或分化。本项目以"物理信号诱导肿瘤干细胞Sox-2表达的表观遗传调控机制"为核心问题，以肿瘤干细胞为研究对象，以Sox-2为切入点，拟采用不同物理机械力刺激，首先研究其对Sox-2表达及H3K9甲基化的影响，并验证Sox-2对自我更新的重要性；其次通过ChIP法及甲基化调控酶抑制剂，研究H3K9甲基化对Sox-2表达与自我更新的调控作用与机制；最终优化物理信号，并验证H3K9甲基化对优化培养的肿瘤干细胞体内自我更新的影响。本项目以物理信号为特色，为肿瘤干细胞表观遗传研究热点提供新的思路和干预新策略。

肿瘤干细胞的存在是肿瘤复发、转移最终导致患者死亡的主要原因之一。在本研究中，我们利用独创的软纤维蛋白与凝血酶反应的细胞培养三维体系，将黑色素瘤及结肠癌肿瘤细胞进行三维培养（3D），发现5天后肿瘤细胞形成克隆球，其数目与大小随基质硬度的增加逐渐减少。与普通二维培养肿瘤细胞（2D）相比，肿瘤干细胞标志物均表达上调，明星信号通路也有显著差异改变。进一步发现，新型抑癌基因DAB2IP在黑色素瘤以及结肠癌干细胞中表达均明显下调，但具体机制不明。我们首先检测了细胞表面力学信号感受器整合素的表达，发现3D黑色素瘤干细胞中ITGB3中表达明显高于普通2D黑色素瘤。其次，利用ITGB3 siRNA转染普通黑色素瘤细胞发现，一方面采用三维培养后3D细胞克隆球大小和数目明显减少，另一方面普通2D黑色素瘤细胞增殖、侵袭和迁移减弱且伴有DAB2IP表达上调。此后，我们又利用DAB2IP siRNA转染普通2D黑色素瘤细胞，一方面采用三维培养发现克隆球大小和数目增多，另一方面发现转染后2D黑色素瘤细胞的增殖、侵袭和迁移能力增强且ITGB3表达回升。最后，构建DAB2IP过表达载体转染普通黑色素瘤细胞后进行三维培养，发现克隆球大小和数目明显减少；转染后2D黑色素瘤细胞的增殖、侵袭和迁移能力被抑制，ITGB3表达下调。上述研究结果表明：黑色素瘤干细胞中细胞表面整合素ITGB3与DAB2IP相互影响调控干细胞特性。此外，我们研究DAB2IP与干性标志Sox-2的相互关系发现，3D黑色素瘤干细胞Sox-2表达较2D细胞明显升高，甲基转移酶3A（DNMT3A）表达也升高。利用DNMT3A siRNA转染普通2D黑色素瘤细胞，发现Sox-2表达上调，而DAB2IP表达被抑制。基于此，我们将DAB2IP过表达载体转染普通2D黑色素瘤细胞，发现Sox-2表达下调而DNMT3A表达上升；相反，采用DAB2IP siRNA抑制DAB2IP表达，引起DNMT3A表达抑制而Sox-2表达上调。该结果提示：Sox-2通过下调DNMT3A而抑制DAB2IP表达，以维持黑色素瘤干细胞的自我更新。