摘要：

目的 探讨FAT1基因在儿童髓母细胞瘤中表达变化及其对预后的影响。方法 收集2004年1月至2016年12月58例儿童髓母细胞瘤标本，全外显子组测序分析其基因变异位点并行Sanger测序验证，实时荧光定量聚合酶链反应和免疫组化染色检测髓母细胞瘤FAT1表达变化、RNA干扰技术敲减其中FAT1基因的表达，MTS法检测细胞增殖率，Kaplan-Meier法绘制生存曲线，Log-Rank检验比较FAT1基因表达变化对总生存期和生存率的影响，并分析FAT1基因表达变化与各项临床因素之间的关系。结果 （1）8例患儿存在FAT1基因致病性错义突变共7个变异位点（187517703C > G、187521337T > C、187524503A > T、187541859C > T、187584610C > G、187628338G > A、187630615G > A），均通过Sanger测序验证。（2）髓母细胞瘤FAT1表达水平低于正常脑组织（P=0.012）。（3）2个靶点（shFAT1和shFAT2）的FAT1基因敲减率均>70%；敲减FAT1基因表达后，髓母细胞瘤细胞增殖率明显增加（均P<0.01）。（4）髓母细胞瘤细胞FAT1低表达组（FAT1阳性细胞比例≤ 50%，38例）患儿总生存期短于（P=0.009）、生存率低于（P=0.009）高表达组（FAT1阳性细胞比例> 50%，20例）。（5）FAT1低表达组患儿年龄< 3岁比例（P=0.037）、肿瘤囊性变比例（P=0.002）高于高表达组；而性别、肿瘤钙化、Chang氏肿瘤分期组间差异无统计学意义（均P>0.05）。结论 FAT1基因呈低表达的髓母细胞瘤患儿预后较差，FAT1基因表达变化可预测髓母细胞瘤患儿预后。

关键词: 髓母细胞瘤, 儿童, 基因, 预后, 细胞, 培养的

Abstract:

Objective To detect the expression of FAT1 gene in children with medulloblastoma and analyze the role of FAT1 gene in prognosis. Methods A total of 58 cases with medulloblastoma were collected from January 2004 to December 2016. The mutation in FAT1 gene was determined by whole exome sequencing (WES) and confirmed by Sanger sequencing. The expression of FAT1 in medulloblastoma tissues was determined by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) technology and immunohistochemistry assay. RNA interference (RNAi) technology was used to reduce FAT1 gene expression in tumor cells and cell viability was detected by MTS method. Kaplan-Meier analysis was used to draw survival curve. Log-Rank test was used to assess the association of FAT1 gene expression with overall survival (OS) and survival rate and clinical factors of different groups of expression. Results 1) WES indicated 7 missense mutations in 8 tissues including 187517703C > G, 187521337T > C, 187524503A > T, 187541859C > T, 187584610C > G, 187628338G > A, 187630615G > A, and the mutations were confirmed by Sanger sequencing. 2) By real-time FQ-PCR assay and immunohistochemistry, the expression of FAT1 in tumor tissues was significantly lower than in normal brain tissue (P=0.012). 3) The knockdown efficiency of two targets (shFAT1 and shFAT2) was over 70%. Cell proliferation rate was increased significantly after FAT1 gene knockdown (P<0.01). 4) Based on the expression of FAT1 by immunohistochemistry, 20 cases demonstrated high FAT1 expression (positive staining >50%) and 38 cases demonstrated low FAT1 expression (positive staining ≤ 50%). The OS and survival rate of patients with low FAT1 expression was significantly shorter than high FAT1 expression group (P=0.009, for all). 5) The ratio of patients less than 3-years old (P=0.037) and cystic change (P=0.002) in low FAT1 patients was higher than the control. But no difference was found in sex, tumor calcification and Chang's tumor classification. Conclusions Children with low FAT1 gene expression displayed worse prognosis. FAT1 gene expression could predict the prognosis of patient.

Key words: Medulloblastoma, Child, Genes, Prognosis, Cells, cultures