AMBRA1介导IGFBP2诱导自噬负向调节骨肉瘤对顺铂敏感性的作用研究

Cisplatin resistance may affect treatment effect of osteosarcoma. Autophagy is reported to be related to cisplastin resistance. Our preliminary study showed expression of IGFBP2 was positively correlated with expression of autohphagic related protein AMBRA1 in osteosarcoma tissue. IGFBP2 was able to induce autophagy in osteosarcoma cell lines. Expression of IGFBP2 was detected in nuclei. And interaction between IGFBP2 and p65 was proved. As a result ,we presume that IGFBP2 enter nuclei of osteosarcoma cells and combine with p65, which is binding with promoter of AMBRA1 gene, and upregulate expressin of AMBRA1 gene. As a result, autophagy is induced, which may lead to cisplatin resistance. The present study is planed to upregualte or downregulate expression of IGFBP2, p65 and AMBRA1, and observe changes of autophagy, in order to study the role of IGFBP2-p65-AMBRA1 pathway in regulating autophagy. CCK8 and FCM experiments are used to detect changes of cisplatin sensitivity when level of autophagy is changed by IGFBP2. ChIP and dual luciferase experiments are used to detect the role of cotransfactor of IGFBP2 in regulating expression of AMBRA1 gene together with p65. Animal experiment is used to confirm the role of IGFBP2 in upregulating AMBRA1 and inducing autophagy, which lead to cisplatin resistance, in vivo. The present study is going to provide new mechanism for cisplatin resistance in osteosarcoma. IGFBP2 may serve as a new therapeutic target of osteosarcoma.

顺铂耐药影响骨肉瘤治疗效果，自噬与顺铂耐药有关。前期研究发现，骨肉瘤组织IGFBP2与自噬相关蛋白AMBRA1表达正相关，骨肉瘤细胞IGFBP2诱导自噬。IGFBP2在细胞核内表达，并可与p65相互结合。我们推测，骨肉瘤中IGFBP2入核，与结合于AMBRA1启动子的转录因子p65结合，上调AMBRA1表达，从而促进细胞自噬，导致顺铂耐药。本研究拟在骨肉瘤细胞中通过依次改变IGFBP2、p65及AMBRA1的表达，观察自噬水平的变化，探讨IGFBP2-p65-AMBRA1通路对自噬的调控；通过细胞活性及凋亡检测，探讨IGFBP2诱导自噬对顺铂敏感性的影响；通过ChIP、双荧光素酶报告实验，探讨IGFBP2在核内辅助p65，调控AMBRA1表达的作用；动物实验验证IGFBP2调控AMBRA1诱导自噬及对顺铂耐药的作用。本研究为骨肉瘤耐药机制提供新的理论依据，IGFBP2可成为骨肉瘤治疗靶标。

IGFBP2在多种肿瘤组织中高表达，是影响肿瘤发生发展的重要因子，并参与肿瘤耐药。自噬的细胞保护作用可能影响肿瘤细胞耐药的重要机制。本研究发现IGFBP2在骨肉瘤组织中的表达高于良性骨肿瘤，高表达IGFBP2与骨肉瘤患者预后不佳相关。骨肉瘤组织中IGFBP2表达与LC3表达正相关。化疗耐药患者IGFBP2的表达高于化疗敏感患者。通过构建IGFPB2过表达和IGFBP2敲减骨肉瘤细胞，探讨IGFBP2对骨肉瘤细胞侵袭能力、自噬及顺铂敏感性的影响。IGFBP2使骨肉瘤细胞侵袭性增强，克隆形成能力增强，提示IGFBP2具有促进骨肉瘤细胞侵袭性的作用。IGFBP2促进骨肉瘤细胞自噬溶酶体数量增多，提示IGFBP2具有诱导骨肉瘤细胞自噬的作用。IGFBP2使骨肉瘤细胞对顺铂敏感性减弱，自噬抑制剂3-MA预处理后的IGFBP2过表达骨肉瘤对顺铂敏感性恢复，提示IGFBP2可能通过诱导自噬参与骨肉瘤细胞耐药作用。Western blot检测显示过表达IGFBP2骨肉瘤细胞p65、HMGB2、Ambra1表达上调，敲减IGFBP2骨肉瘤细胞p65、HMGB2、Ambra1表达下调，提示IGFBP2促进骨肉瘤细胞侵袭性、诱导自噬及顺铂敏感性减弱等作用可能通过HMGB2、Ambra1、NF-κB信号通路起作用。本研究提示IGFBP2在骨肉瘤发展中的作用，并对顺铂敏感性的影响作用，IGFPB2可作为骨肉瘤治疗的靶标，抑制骨肉瘤的侵袭性、调节顺铂敏感性，提高骨肉瘤患者生存率和生存质量。