4.1N对尤文肉瘤恶性行为的抑制作用及其机制研究

Ewing sarcoma is one of the most frequent malignant tumors in children and adolescents. Ewing sarcoma are characterized by early recurrent or metastases with the 5 year survival less than 30%. However, the mechanism of influence on the malignant behaviour in Ewing sarcoma is still unknown. The applicant found firstly that 4.1N play an important role in inducing cellular differentiation through 4.1N gene knock-out mice model. Furthermore, a significantly lower expression even deletion of 4.1N in Ewing sarcoma tissue and cell lines was observed. The expression of 4.1N was inversely related with that of Ki-67 and p-AKt, whereas the expression of 4.1N was strongly associated with cellular differentiation. In 4.1N positive cell line, nerve growth factor (NGF) treatment leads to the translocation of 4.1N expression into the nucleus, which prevent the expression of p-Akt and cell proliferation. This study will detect systematically the status of 4.1N loss of heterozygosity (LOH), gene mutation and methylation in Ewing sarcoma samples by microsatellite analysis and methylation specific PCR. The influence of 4.1N on the malignant behaviour in Ewing sarcoma cell lines were observed by gene transfer and knock-down methods. The correlation of 4.1N with PI3K/Akt signal pathway will also be investigated. The aim of this study is to confirm the inhibition effect together with its mechanism of 4.1N on the malignant behaviour in Ewing sarcoma, in order to provide a new marker for the evaluation of prognosis and a new idea for the molecular targeted therapy in the tumor.

尤文肉瘤是儿童和青少年常见恶性肿瘤之一,早期即会发生复发或转移、且5年生存率小于30%，但影响其恶性行为的机制尚不清楚。申请者通过4.1N基因敲除小鼠首次发现4.1N具有促进细胞分化的作用。进一步研究还发现：4.1N在尤文肉瘤组织和细胞系中的表达明显下降、甚至缺失；4.1N低表达与Ki-67和p-AKt高表达呈负相关，而与细胞分化程度呈正相关；神经生长因子作用于4.1N阳性尤文肉瘤细胞，使其表达转至细胞核、并抑制p-Akt的表达。本课题拟：采用微卫星分析和甲基化特异性PCR方法查明尤文肉瘤病例中4.1N基因杂合缺失、突变和甲基化的状况；通过将4.1N基因导入和敲除技术查明4.1N对尤文肉瘤细胞恶性行为的影响程度；探索4.1N和PI3K/Akt通路相互作用的机制。研究结果将有助于揭示4.1N在尤文肉瘤恶性行为中的作用及其机制，进而为尤文肉瘤的预后判断提供新标志物和为靶向治疗提供新思路。

尤文肉瘤恶性程度高、进展快,早期即可发生转移，其恶性生物学行为是影响患者预后和治疗的重要因素。4.1N(EPB41L1)基因属于4.1家族成员之一，前期研究发现4.1N在尤文肉瘤中主要呈低表达及缺失，并可能在肿瘤发展演进中与其它信号分子协同发挥抑癌作用，但4.1N基因失活及其作用机制未明。本项目通过系统研究发现尤文肉瘤中4.1N基因存在显著微卫星不稳定性（MSI)，其中D20S174位点MSI发生频率最高，其次为D20S884、D20S478及D20S908位点，并主要见于4.1N低表达和缺失肿瘤组织。我们还发现4.1N基因exon2和exon12在尤文肉瘤中出现杂合及纯合突变，其中exon12突变率为43%；伴有MSI的病例其4.1N基因突变频率明显增高；同时出现D20S478位点MSI和exon2／exon12突变以及D20S174位点MSI和exon12突变的病例预后较差。我们首次通过高通量测序在4.1N缺失和低表达尤文肉瘤中检测到PIK3CA、LKB1、APC、PTPN11、EGFR、KDR基因改变，其中LKB1与肿瘤细胞增殖明显相关；而 4.1N高表达病例则主要检测到MLH1、KDR基因改变。细胞生物学实验发现4.1N基因沉默可影响尤文肉瘤细胞的生长速度及其分化，PI3K/Akt信号通路相关分子periostin表达下调可升高4.1N蛋白表达，Periostin表达与患者预后差有关；进一步研究发现periostin 及分化相关分子Id2对于尤文肉瘤细胞生长、侵袭等恶性生物学行为具有重要影响。这些研究结果表明，4.1N基因在尤文肉瘤中的失活机制主要涉及MSI及突变所引起的基因组不稳定性；4.1N蛋白可能通过影响 periostin发挥肿瘤抑制作用；Periostin和LKB1可作为新的判断尤文肉瘤生物学行为的分子标志物。