AXL通过外泌体Linc00852-miRNA-AXLmRNA正反馈调控环路介导不同转移潜能细胞间通讯在骨肉瘤转移中的作用及机制研究

It is very common for osteosarcoma to have early distant invasion and metastasis. Understanding the underlying mechanisms and developing effective strategies against lung metastasis of osteosarcoma are highly desired in the clinic. Recently, exosome transmitted as intercellular signal to regulate tumor invasion and metastasis is becoming the focus of tumor research. We have identified that there were spatial heterogeneity of the expression of receptor tyrosine kinase AXL in osteosarcoma tissues through the immunohistochemistry staining. Also the osteosarcoma cell lines with highly expression of AXL protein could produce exosomes, which promoted the capability of invasion and metastasis of AXL lower expression cell lines invitro. High-throughput sequencing of lncRNA showed that linc00852 was obviously highly expressed in the exosomes created by AXL highly expression osteosarcoma cells. Consistently, the expression of linc00852 in osteosarcoma tissues were significantly higher than that in corresponding normal soft tissues. All the data indicated that exosome linc00852 may practice as an intercellular signal transferd from AXL high expression osteosarcoma cells to AXL low expression cells, which may up-regulate the AXL mRNA to promote the invasion and metastasis of osteosarcoma with the molecular mechanisms unclear. Here we will prove the intercellular regulation by paracrine secretion of exosome linc00852 among osteosarcoma cells, and discover the mechanism of linc00852 enwrapped in exosome, also announce the role of linc00852 as a ceRNA competing with miRNA to control the transcription of AXL mRNA and the sequencing AKT signal pathway. The clinical significance of exosome linc00852 will be confirmed in the osteosarcoma tissues and the patients’ serum. This work will bring light to identify the paracrine secretive regulation of exosome linc00852 during the process of the invasion and metastasis of osteosarcoma, in order to find the potential therapeutic target of osteosarcoma.

骨肉瘤(OS)转移是患者主要死因。我们前期研究发现受体酪氨酸激酶AXL促进OS侵袭转移，并具有转移潜能相关异质性；AXL高表达细胞株产生的外泌体促进低表达株迁移侵袭，高通量测序及验证发现AXL高表达株外泌体linc00852显著上调；OS细胞中linc00852可能竞争性结合miR-7-5p而上调AXLmRNA水平；因此我们推测AXL可能通过外泌体linc00852-miRNA-AXLmRNA正反馈调控环路传递不同转移潜能OS细胞间调控信息，而促进肿瘤转移。本研究拟通过体内外实验证明linc00852通过外泌体传递并反馈上调AXL以促进OS转移；应用RIP,RNApulldown等鉴定linc00852包裹入外泌体及其发挥ceRNA作用调控AXL-AKT信号分子的机制；并在病例中验证外泌体linc00852的临床意义；从而阐明细胞间旁分泌正反馈调控促进OS转移机制，探索OS潜在治疗靶标。

骨肉瘤是好发于青少年的最常见恶性骨肿瘤，其复发和转移率高，社会危害性大。本项目在前期研究的基础上进行，课题组前期发现受体酪氨酸激酶AXL在骨肉瘤细胞中高表达，并与肿瘤进展密切相关；骨肉瘤组织中存在不同AXL表达水平的细胞族群，其血管侵犯和远处转移能力明显不同。在本项目的研究中，我们发现AXL高表达细胞产生的外泌体内AXLmRNA和蛋白水平显著高于AXL低表达细胞，且AXL高表达外泌体与AXL低表达细胞共培养后能促进AXLmRNA和蛋白表达，并促进AXL低表达细胞体内和体内外增殖及侵袭；高通量测序分析发现两类外泌体中Linc00852的表达差异最大，因此外泌体介导的Linc00852可能是促进骨肉瘤增殖和侵袭转移的重要因子。应用慢病毒稳转上调Linc00852后骨肉瘤细胞的体内外增殖、迁移、侵袭能力明显增强；且肿瘤组织中Linc00852的高表达与骨肉瘤患者的复发和转移呈显著性正相关；FISH检测发现Linc00852主要表达于骨肉瘤细胞核内，应用RNApulldown和质谱筛选出骨肉瘤细胞内与Linc00852最重要的互作蛋白为RNA结合蛋白PTBP1，慢病毒转染过表达linc00852后骨肉瘤细胞中PTBP1和AXL的mRNA表达水平明显升高；而应用shRNA 敲低linc00852表达后骨肉瘤细胞中PTBP1和AXLmRNA和蛋白表达明显降低；多株骨肉瘤细胞中PTBP1表达水平明显升高，并通过促进AKT的活化而促进OS细胞体外和裸鼠体内的增殖和侵袭，PTBP1和p-AXL共表达是影响骨肉瘤患者总生存率的独立影响因素。细胞回复实验证明linc00852和PTBP1的共表达促进AXL表达的升高；PTBP1对AXL表达的调控是通过选择性剪接AXLmRNA形成活化的AXL-S而实现的；这些研究结果提示不同的细胞族群之间存在AXL/Exosome-Linc00852/Linc00852/PTBP1/AXL/AKT/AXL调控环路，这一发现对于认识骨肉瘤细胞侵袭转移的分子机制具有重要意义，而且鉴于其他肿瘤如肺癌中AXL的靶向治疗已经开始应用于临床研究，因此本研究为骨肉瘤中针对AXL的靶向治疗提供了重要的理论依据。