Snf5介导的表观修饰调控在MSC自发转化中作用机制的研究

Accumulating data have shown that mesenchymal stem cells (MSCs) from various species frequently undergo spontaneous transformation after long-term culture. The transformed MSCs demonstrate some characteristics of malignant cells and can form fibrosarcoma and osteosarcoma when injected into immunodeficient mice. Recent studies have revealed that p16INK4a is silenced by the epigenetic modifications in transformed MSCs, but the epigenetic mechanisms in the stable silence of p16INK4a gene remain unclear. This study is designed to determine the role of Snf5, an epigenetic regulatory factor, during the spontaneous transformation of MSCs by RNA interference and exogenous expression, especially in the switch between the reversible suppression and stable silence of p16INK4a gene by bisulphate sequencing and chromatin immunoprecipitation assay. The epigenomic modifications controlled by these regulatory mechanisms will be evaluated by the high-throughput microarrays. Furthermore, the meanings of relieving the stable silence will be verified by exogenous expression of Snf5. Our research could reveal epigenetic mechanism in the spontaneous transformation of MSCs and tumorgenesis. The study will be of great scientific significance in the diagnosis and treatment of tumors.

间充质干细胞(mesenchymal stem cell，MSC)在体外长期培养过程中会发生自发转化，转化后的MSC在移植到裸鼠体内后具有形成纤维肉瘤和骨肉瘤的能力。本课题组及国外研究均发现：由表观遗传学修饰模式改变引起的p16INK4a基因的稳固沉默在MSC自发转化中起重要作用。为分析p16INK4a基因的表观遗传修饰模式发生改变的机制，本项目以表观遗传修饰调控蛋白Snf5为切入点，采用RNAi和外源性表达的方法分析Snf5在p16INK4a基因的可调控抑制和稳固沉默模式相互转换中的作用；通过高通量芯片技术分析该机制是否同时参与其它抑癌基因的表观遗传学沉默；并进一步探讨恢复Snf5表达能否解除这种稳固沉默模式，甚至逆转自发转化的过程。该研究能为MSC自发转化和肿瘤发生提供表观遗传学理论依据，在肿瘤的诊断和治疗以及转化医学中具有重要的科学意义和潜在的应用价值。

间充质干细胞(mesenchymal stem cell，MSC)在体外经过长期培养后会发生自发恶性转化，这种转化后的MSC在移植到裸鼠体内后具有形成肉瘤的能力。在本项目通过连续传代培养的方式构建了MSC自发转化模型，通过细胞计数和软琼脂集落形成实验发现转化后MSC具有更强的增殖能力并且获得了非锚定生长的能力；为了研究p16INK4a基因稳固沉默的表观遗传学机制，通过染色质免疫共沉淀、亚硫酸盐修饰后测序、RNA干扰和外源性表达等技术分析了Snf5与p16INK4a表达的关系，特别是Snf5对p16INK4a基因表观遗传学修饰模式的影响。在转化后MSC，伴随p16INK4a基因的表达降低，Snf5的表达明显降低；通过对转化后MSC外源性表达Snf5能够增加p16INK4a基因H3k4me3的修饰水平，在一定程度上恢复p16INK4a的表达，但对p16INK4a基因的DNA高甲基化状态没有影响。通过对衰老前MSC干扰Snf5的表达后，p16INK4a基因的H3K4me3修饰水平降低，DNA甲基化水平升高，同时p16INK4a基因的表达降低；干扰Snf5后，衰老前MSC的增殖能力下降。这说明，Snf5在调控p16INK4a的表达中起重要作用，Snf5对p16INK4a的调控参与到MSC自发转化过程，然而MSC自发转化还需要其他分子机制的参与。