TIM-3 促上皮-间质转化诱导骨肉瘤侵袭及转移的分子机制研究

Osteosarcoma （OS） is one of the serious carcinoma characterized by malignant invasion and metastasis that harm to human health. Nevertheless,the mechanism that induces tumor cell invasion and metastasis is unclear. Our previous work has found that the novel immunosuppressive molecule, T cell immunoglobulin and mucin-domain-containing molecule-3 (TIM-3),was specific expression in human osteosarcoma tissue. Interestingly, it co-expressed with some epithelial-mesenchymal transition (EMT) markers like Ecad, Smad, Snail et al., suggesting that TIM-3 should promote the pathogenesis of osteosarcoma via inducing the EMT process (Oncol Lett. 2013;6:490-494). In this project, the expression of TIM-3 in the osteosarcoma cell line-MG-63 cells should be overexpressed by DNA transfected technique, and, the capacity of tumor cell invasion should be analyzed by transwell experiments ex vivo. Moreover, TIM-3-MG63 and the control cells should be transferred into the SCID mouse to establish an xenograft tumor model, the tumor size, mouse survival, the EMT process and other parameters should be compared and analzyed, these tudies should further confirm that TIM-3 can enhance tumor metastasis through EMT in vivo. Finally, a cohort of clinical samples of OS shoule be cellected from our department and the expression of Tim-3 should be detected by immunohistochemistry, therefore, the relationships between Tim-3 expression and patients survival rate (survival times) should be analyzed.To measure how Tim-3 signal control EMT progress, overexpression of Tim-3 in MG-63 cells and the expression of EMT related molecules, including ESRP, FGFR2, N-cad, Slug et al should be analyzed by western-blot, qPCR and EMSA. In a word, this research will elucidate the molecular mechanism of Tim-3 promoting metastasis of laryngeal cancer, which might help us develop some novel methods for early diagnosis and treatment of OS.

前期研究我们发现免疫分子Tim-3特异表达于人骨肉瘤组织并与上皮-间质转化(EMT)标志物共表达，提示Tim-3可能通过诱导EMT促骨肉瘤的侵袭及转移（Oncol Lett. 2013:490-494）。然而Tim-3促进EMT的分子机制尚未明确。本研究拟收集大样本量的骨肉瘤临床标本，分析Tim-3表达与骨肉瘤术后预后的相关性；在人骨肉瘤MG-63细胞表达Tim-3，采用qPCR、EMSA及Western-blot等技术分析Tim-3信号下调ESRP/FGFL2b导致EMT进程的分子机制；并应用划伤愈合及Transwell培养技术分析TIM-3对细胞侵袭能力的影响；最后建立人MG-63细胞的SCID小鼠移植瘤模型，对比肿瘤大小、小鼠存活及EMT进程等指标，证实TIM-3可通过EMT作用增强癌细胞转移。本研究将阐明Tim-3促骨肉瘤转移的分子机制，对骨肉瘤的早期诊断及临床治疗有重要意义。

骨肉瘤（osteosarcoma，OS）是一种起源于骨间叶细胞的原发性恶性骨肿瘤。典型骨肉瘤临床少见，其发病率约为0.3/万，约占恶性肿瘤的0.2%，原发骨肿瘤的15%。骨肉瘤好发于男性青少年，恶性程度高，侵袭性强，早期易发生肺转移，预后较差，以往单一治疗疗效差，５年生存率不到20％。因此，对骨肉瘤细胞侵袭和转移机制的研究是有效治疗的核心问题，对有效控制复发、提高患者的生存率至关重要。上皮-间质转化（epithelialmesenchym al transition，EMT）在肿瘤侵袭和转移有重要作用。Ｔ细胞免疫球蛋白黏蛋白-3（TIM-3）基因是人类TIM 基因家族之一，特异性地表达于人和小鼠的Th1 细胞表面，负调节Th1细胞免疫应答。然而, TIM-3是否可通过诱导骨肉瘤的EMT进程促进喉肿瘤的转移尚未知。. 课题受资助以来重要的研究结果包括：（1）免疫组化我们发现共刺激分子TIM-3表达与骨肉瘤组织中。 (2）免疫双标显示TIM-3表达于多种细胞表面，包括PCNA+ 的增值性肿瘤细胞、 Bcl-2+ 抗凋亡肿瘤细胞中，此外，其也表达于CK-18+ 上皮细胞、CD31+内皮细胞及CD68+ 巨噬细胞中。 （3） 双荧光显色提示TIM-3与EMT标志物如Vimentin、pSmad2/3及Slug等标志物共聚与同一细胞，提示B7-H3可能参与EMT的过程。（4）使用RNAi技术可特异性干扰骨肉瘤细胞系MG-63细胞TIM-3表达 。（5）干扰TIM-3表达可抑制TGF-β诱导的EMT进程，如MG-63细胞表达EMT相关分子包括Vimentin、Snail、Slug在TIM-3受干扰后下调表达。提示TIM-3 可促进EMT进程。(6)分子机制研究Tim-3可激活下游的syk-pkc信号通路，对骨肉瘤发生的促进作用，文中目前正在投稿中。(7)在课题的资助下，我们系统研究EMT诱导喉癌发生及发展的机制，发表了相关的SCI论著三篇。