miR-29与TGF-β调控网络对滑膜肉瘤干细胞生物学特性的影响及侵袭转移机制的研究

Synovial sarcoma (SS) is a highly aggressive sarcoma of the soft tissue with a strong potential to metastasize and characterized by translocations encoding SYT-SSX fusion genes. Its histological origin and pathogenesis are uncertain, which is hypothesized to be related to cancer stem cells (CSC). One of the present study focuses is that miRNAs regulating CSC properties have been shown to play a role in metastasis, involving TGF-β1 and other pathways. Our studies have shown that TGF-β1 regulates SYT-SSX1 promote EMT process and the cancer stemness that induce the ability of increased invasion and migration. Our previous exon chip detection and bioinformatics analysis have found significantly miR-29 which target genes associated with TGF-β1. Based on these observations, this project aims at detecting the expression of miR-29 and CSC related genes in the subtypes and cellular components of synovial sarcoma in tissues. We will construct SYT-SSX1 high expression cells in vitro using a mimics and siRNA miR-29 to treat study cells, and then make sphere formation to analyze possible correlations between TGF-β1 and miR-29 by treating with TGF-β1 or an inhibitor. In nude mice studies, tumor metastasis, proliferation and apoptosis of cells in each group will be detected and analyzed. Therefore, the completion of this project may have important biological significance and potential clinical applications.

滑膜肉瘤（SS）是具有特异性SYT-SSX融合基因和高侵袭和转移的软组织肉瘤。迄今为止其起源和发病机制仍未不清，近年有文献提示SS可能与肿瘤干细胞（CSC）有关，课题组前期研究结合miRNA调控CSC影响肿瘤侵袭转移的热点研究方向，初步证实TGF-β1可诱导SYT-SSX1促进EMT和瘤细胞干性特征，导致侵袭和迁移能力增加，芯片结果筛选出差异miR-29存在与TGF-β通路相关的靶基因。基于此，本项目预从滑膜肉瘤组织检测不同类型及成分中miR-29和CSC相关的表达，体外实验中，构建SYT-SSX1高表达的细胞，运用模拟物及siRNA建立miR-29高/低表达细胞，进行干细胞成球实验，进一步用TGF-β干预，分析和检测各组中TGF-β、miR-29、CSC相关性；通过裸鼠成瘤实验检测各组肿瘤转移、增殖、凋亡情况。本项目将为临床治疗SS积累基础数据，因此具有重要的理论研究意义和临床应用价值。

目的：滑膜肉瘤（SS）是具有SYT-SSX融合基因和高侵袭及转移性的软组织肉瘤，近年有学者认为SS可能与肿瘤干细胞（CSC）有关，本研究初步证实miR-29与TGF-β通路下游基因EP300 存在靶向关系，并对SS的CSC、增殖及侵袭迁移具有明显影响，为进一步研究滑膜肉瘤肿瘤干细胞靶向治疗提供一定理论基础。.结果：①通过无血清悬浮方法培养后，发现SYT-SSX1型高表达细胞比SW982成球能力高，且qRT-PCR、Western-Blot、流式细胞仪及生物学功能检测显示，SYT-SSX1细胞 Sphere细胞具有干性特征。②重组剂TGF-β1处理SYT-SSX1 Sphere细胞后, Sphere细胞数目、体积明显增加且干性标志物表达也明显增强；反之，抑制剂SB431542处理后, Sphere细胞数目及体积明显减少，且Sphere细胞干性标志物也明显减弱。③ 生物信息学分析显示TGF-β1下游基因EP300是miR-29b的靶基因，并且EP300与miR-29b-5p具有结合位点。双荧光素酶报告基因和RIP实验结果表明：miR-29b-5p与EP300有靶向关系，在细胞内直接结合，并且发挥抑制EP300的作用。miR-29b-5p在滑膜肉瘤组织及细胞中的表达与EP300呈负相关。转染miR-29b-5p mimic后，TGF-β1及p-Smad2/3表达下降，细胞成球能力、sphere细胞 CSC 标记物表达、球体细胞的增殖、侵袭迁移能力均降低，相反，转染siRNA-29b-5p后，TGF-β1及p-Smad2/3表达升高，细胞成球能力、sphere细胞 CSC 标记物表达、球体细胞的增殖、侵袭迁移能力均升高。且miR-29b均受到EP300 的调控。④通过Arraystar circleRNA芯片技术在SS组织中筛选13个差异circRNAs，其中hsa_circ_0000518与滑膜肉瘤患者的不良预后密切相关。.结论：SS细胞具有肿瘤干细胞样特征，SYT-SSX1 细胞CSC特征更强；miR-29b 靶向调控 EP300 抑制 SS 球体细胞的干细胞样特征及增殖、侵袭迁移能力；差异circRNAs可能参与SS的发生发展。