利用严密型Tet-on系统重建INI1表达诱导上皮样肉瘤定向分化及其表观遗传学机制研究

Epithelioid sarcoma (ES) is a soft tissue original highly invasive malignant tumor. The tumor cells possess the epithelial and mesenchymal differentiation potential. The clinical metastatic and recurrent rates are extremely high, while the prognosis is poor. Integrase interactor l (INI1) is a core subunit of human SWI/SNF remodeling complexes, plays a critical role in epigenetic regulation of tumorigenicity and differentiation and loss of INI1expression is the molecular feature of ES. In our preliminary study, we found that using a strict Tet-on system to re-express INI1 could induce the epithelioid sarcoma cells directly differentiated to adipocyte, however, the in vivo effect and the underlying epigenetic mechanism is still unclear. In the present project, we would like to confirm the directed differentiation induced by INI1 in vitro and in vivo; Chip-chip, cDNA microarray and bioinformatics analysis are performed to screening the downstream target gene of INI1; moreover, chip, luciferase report assay, western blot and DNaseⅠhypersensitivity analysis are used to validate those INI1target genes; finally, the function and clinical value of these target genes are also assessed. Taken together, this project aim to assess the value of differentiation therapy targeting INI1 in ES, screen the target genes of INI1, elucidate the possible pathogenesis of ES, seek the novel therapeutic target to enriches the treatment strategies of ES and improve the whole clinical outcomes.

上皮样肉瘤（ES）是一种软组织来源恶性肿瘤，具有上皮及间质分化潜能，临床转移、复发率高，预后差。整合酶相互作用分子1（INI1）是ATP依赖染色质重塑复合物SWI/SNF核心亚基之一，在细胞分化及成瘤中发挥重要表观遗传学调控作用。我们前期研究表明INI1缺失是ES标志性改变,进一步发现严密型Tet-on系统重建INI1表达可诱导ES细胞向脂肪细胞分化，但其体内诱导效应及表观遗传学调控机制尚未明确。现拟结合前期基础进一步验证INI1体内诱导ES向脂肪细胞分化的作用；通过ChIP-chip、表达谱芯片结合生物信息分析筛选INI1靶基因；利用chip、荧光素酶报告基因检测及DNaseⅠ高敏性分析等方法验证相关靶基因；结合功能实验及临床病例分析靶基因功能及临床意义。本研究旨在探讨INI1诱导分化在ES治疗中的作用，明确其通过染色质重塑机制调控的靶基因群，寻找ES新的治疗靶标，进而改善ES治疗效果

上皮样肉瘤（ES）是一种软组织来源恶性肿瘤，具有上皮及间质分化潜能，临床转移、复发率高，预后差。整合酶相互作用分子1（INI1）是ATP依赖染色质重塑复合物SWI/SNF核心亚基之一，在细胞分化及成瘤中发挥重要表观遗传学调控作用。本课题研究通过严密型Tet-on系统重建INI1表达，研究其在诱导ES细胞分化中的作用及相关的分子调控机制。研究结果表明通过严密型Tet-on系统重建INI1表达在动物体内外均可诱导ES细胞向脂肪细胞定向分化，发现在上皮样肉瘤中存在多梳蛋白家族成员EZH2过表达现象，同时我们发现INI1诱导表达后可显著抑制EZH2表达量，H3K27甲基化水平显著下调，与此同时调控脂肪分化的关键调控因子PPAR-γ 及CEBP-α 表达上调，从而促进上皮样肉瘤细胞向脂肪向分化。本研究为上皮样肉瘤诱导分化治疗提供新的分子靶标，为改善上皮样肉瘤的整体临床效果提供理论依据及实验基础。