TGF-β1通路调控MET在滑膜肉瘤双相分化和侵袭转移中作用及机制

Epithelial-mesenchymal transition (EMT/MET) is closely associated with the invasion and metastasis of cancer, and involved in the regulation of TGF-β1 signaling pathway and transcription factors. On the other hand, TGF-β1 is also an important inducer of EMT. However, the role of EMT/MET in sarcoma progression is considerably less well understood. Synovial sarcoma (SS) is a malignant tumor of the soft tissue characterized by uncertain histological origin and has biphasic morphology of mesenchyme and epithelium. Clinical outcomes of SS are very poor and approximately 90% of patients die from metastatic disease. The biphasic morphology and high invasiveness of SS make it a good model for investigating the relationship between TGF-β1-induced EMT/MET and fusion gene SYT-SSX. The study will then separate epithelium and mesenchyme from SS tissues using laser microdissection technique; analyze the expression of TGF-β1 and EMT related markers by RT-PCR; and detect genomic alterations using CGH or Array CGH technique, respectively. This study will first analyze the expression of TGF-β1 pathway and EMT related markers, and fusion gene SYT-SSX in the epithelium and mesenchyme from SS tissues using laser microdissection technique. Furthermore, we will use a specific TGF-β1 inhibitor or inducer to treat SS cells and examine the changes of EMT-related markers as well as the invasion capability and reproductive activity of treated cells. This study may reveal that TGF-β1-regulated EMT renders a functional role in the invasiveness and metastasis of SS that may be correlated the fusion gene SYT-SSX. This research may provide a new target for the treatment and prognosis of SS.

上皮间叶转化（EMT/MET）与肿瘤的侵袭、转移密切相关，涉及TGF-β1等通路和转录因子调控。但间叶源性肿瘤中EMT/MET的研究少见。滑膜肉瘤（SS）是一种组织起源不明、具有上皮和间叶双相分化的软组织肉瘤，90%患者死于转移。SS双相分化的形态特征和高侵袭性的临床经过为系统地探讨TGF-β1介导MET/EMT发生及与融合基因SYT-SSX的关系提供了良好模型。本研究拟在免疫组化等技术检测SS组织和细胞株中TGF-β1通路与MET/EMT相关分子和融合基因表达基础上，运用激光显微切割结合RT-PCR和CGH/ArrayCGH，分别检测SS上皮和间叶两种成分中以上指标表达及染色体基因组改变；在SS细胞株中干预TGF-β1表达，检测干预前后细胞形态及以上指标的变化，探讨TGF-β1通路调控EMT/MET在SS双相分化和侵袭转移中的作用机制及与融合基因的关系，为SS的治疗和预后评估提供新靶点。

研究背景.滑膜肉瘤（SS）是一种组织起源不明、具有上皮和间 叶双相分化的软组织肉瘤，90%患者死于转移。SS双相分化的形态特征和高侵袭性的临床经过为系统地探讨TGF-β1介导MET/EMT发生提供了良好模型。本研究通过免疫组化、激光显微切割、外显子芯片及体外细胞实验等技术，探讨TGF-β1通路调控EMT/MET在SS双相分化和侵袭转移中的作用机制。.研究内容.1.运用免疫组化检测SS组织中TGF-β1和EMT相关蛋白的表达。2. 运用激光显微切割技术分选及提取12例双相型SS组织中的上皮和间叶两种细胞成分，通过外显子芯片检测两种成分的SNP位点且进行功能富集分析。3.在SW982人SS细胞系，将TGF-β1对SW982进行干预，检测EMT相应指标表达及各组细胞迁移、侵袭和增殖能力。4.统计学分析，进行方差分析和t检验等，以P<0.05，差异具统计学意义。.结果.1. 免疫组化及临床病理分析结果显示：1）不同类型SS之间EMT相关因子的表达有差异；2）E-cadherin阳性或阴性表达病例中，smad2/3\、N-cadherin和β-catenin，与E-cadherin表达具有相关性。3）Snail和Smad2/3的表达与临床分期有关。4）E-cadherin阴性的患者预后更差。.2. 外显子芯片结果显示： 1）12例双相型SS中上皮和梭形细胞两种成分差异SNP位点有336个。2）结合WGAVewer及Illumina的注释信息分析，具有显著性差异（P<0.001）有23个基因。3）通过生物信息学分析，这些差异基因涉及细胞粘附、细胞外基质、TGF-β等信号通路。.3. 体外实验结果显示：1）TGF-β1诱导后，E-cadherin的表达降低，N-cadherin和转录因子Snai、Slug的表达显著升高。相反SB431542处理后，E-cadherin表达增加，N-cadherin及转录因子snail和slug均明显降低。2）TGF-β1激动剂处理组细胞侵袭、迁移和增殖能力明显增强；相反抑制剂SB431542处理的细胞侵袭、迁移和增殖能力均下降。.结论.SS上皮和间叶两种成分中存在不同基因组改变，主要与粘附、细胞外基质、TGF-β等通路有关。2. TGF-β1的激活或阻断可调控EMT相关因子的表达，促进或抑制EMT过程，影响SS细胞侵袭、迁移和增殖能力。