自噬相关基因PLEKHM1调控CD70+骨肉瘤起始细胞自我更新与分化的作用及机制研究

The malignance of osteosarcoma with poor prognosis is high. Osteosarcoma stem cells are responsible for clinical treatment failure. Lacking of precise and speicific markers restricts the research process involving in targets and signaling of osteosarcoma stem cells. We detected the sphere cells and resistant cells by microarry, and indentified CD70+ cells have the characterics of CSCs; and expression of the autophagy-relatived gene PLEKHM1 is much higher, the autophagy-lysosome and NF-κB signaling both are activated in CD70+ cells; Knockdown of PLEKHM1 suppresses the characterics of CSCs and reduces the degradation of IκBa. Those suggest that PLEKHM1 may regulate the autophagy-lysosome and maintain the feature of CSCs by activating the NF-κB signaling in osteosarcoma stem cells. The present study aims to study the CD70+ cells using adopt microfluidic chips, flow cytometry;demonstrate the role of PLEKHM1 by lentivirus system; confirm the downstrem sigaling by microarry; disclose the mechnisam of PLEKHM1, autophagy-lysosome and NF-κB signalin using protein MS, IP and TEM. Finally, adopt tissue array will be used to confirm the interactive regulation among components of the pathway and its correlation with clinical prognosis. The present study will confirm precise and speicific marker of osteosarcoma CSCs,bring new understanding and concept to the initiation and progression of osteosarcoma and broken the limitation of current treatment.

骨肉瘤恶性度高，预后差，其干细胞是临床治疗失败的根源。缺乏精准特异标记物制约着骨肉瘤干细胞关键靶点的研究进程。我们前期运用表达谱芯片检测成球及耐药骨肉瘤细胞，验证发现CD70+细胞具有干细胞特性；且CD70+骨肉瘤细胞高表达自噬相关基因PLEKHM1，自噬-溶酶体途径及NF-κB通路异常活化；敲低PLEKHM1削弱骨肉瘤细胞干性，减少IκBa降解。提示PLEKHM1可能通过自噬-溶酶体途径异常活化NF-κB维持骨肉瘤干细胞特性。本研究拟采用单细胞水平检测、流式细胞技术等全面阐明CD70+细胞表型特征；慢病毒双向干预系统等探讨PLEKHM1关键作用；表达谱芯片筛选下游通路，蛋白质谱、IP及透射电镜扫描等揭示PLEKHM1调控自噬-溶酶体及NF-κB活性的机制；组织芯片确认该调控关系及临床意义。本项目的完成，为骨肉瘤干细胞确证出精准标记物；深化了骨肉瘤干细胞的研究，为突破临床治疗瓶颈提供新策略

1.骨肉瘤恶性度高，预后差，肿瘤干细胞是临床治疗失败的根源。缺乏精准特异标记物制约着骨肉瘤干细胞关键靶点的研究进程以及缺乏低毒高效靶向药物而使骨肉瘤治疗停止不前。我们运用表达谱芯片检测成球及耐药骨肉瘤细胞，验证发现CD70+细胞具有干细胞特性；且CD70+骨肉瘤细胞高表达自噬相关基因PLEKHM1，自噬-溶酶体途径及NF-κB通路异常活化；敲低PLEKHM1削弱骨肉瘤细胞干性，减少IκBa降解。提示PLEKHM1可能通过自噬-溶酶体途径异常活化NF-κB维持骨肉瘤干细胞特性，揭示了PLEKHM1调控自噬-溶酶体及NF-κB活性的机制，同时组织芯片明确该调控关系及临床意义。本项目为骨肉瘤干细胞确证出精准标记物；深化了骨肉瘤干细胞的研究，为突破临床治疗瓶颈提供新策略。.2.通过筛选抗骨肉瘤天然化合物，通过体内外一系列研究发现半乳糖替告皂甙（DGT）具有高效、低毒抗骨肉瘤作用。分子机制探讨中，通过对关键调控靶点的筛选，证实半乳糖替告皂苷抑制GSK3β活性后能够下调Hedgehog/Gli1信号通路的转录活性。该研究明确天然活性产物去半乳糖替告皂甙抗骨肉瘤的作用及关键调控靶点，为将半乳糖替告皂甙开发成抗骨肉瘤药物提供理论依据。同时，还能促进我国中医药学抗肿瘤研究事业的发展，从而产生良好的社会效益和经济效益。.3.通过对公共数据库进行分析，结合该项目中的芯片检测结果，研究CDC20及其下游靶基因（securin, cyclin A2 and cyclin B2）作为骨肉瘤预后的分子指标的重要作用。