KIF2B基因突变及失活在肝癌发生发展中的意义

In our preliminary work, using high throughout single nucleotide polymorphism (SNP) chips in a pilot genome-wide association study, we have revealed the direct relationship between rs4561519 (leading to Arg→Gly missense mutation) in coding region of kinesin family member 2B (KIF2B) gene and the occurrence of hepatocellular carcinoma (HCC).In this research, further study will be performed to elucidate the function and mechanism of KIF2B gene mutation in the development of HCC. First, we will validate the relationship between the KIF2B gene mutation with its expression and the occurrence, and prognosis of HCC, via the analyses of the discipline in the genotype and expression of KIF2B in HCC tissue and adjacent non-cancerous liver tissue. Second, we will detect the promoter methylation status by methylation-specific PCR and investigate the ability of 5-aza-2'-deoxycytidine to induce KIF2B gene re-expression and its effects and mechanisms in HCC cell proliferation and apoptosis. Third, we will construct the wild type and mutant type KIF2B expression plasmids and the KIF2B interference plasmid and explore the biological roles of KIF2B over-expression or down-regulation (both wild type and mutant type) in vitro, cell growth, proliferation, apoptosis，and migration. Fourth, tumorigenicity assay in nude mice will be performed via inoculating the stable cell lines. We will analyze the influence of the nude mice's tumor growth when the KIF2B gene expression has been inhibited or enhanced. Finally, we will analyze the interacting proteins with KIF2B in HCC cells. This study will elucidate the function of tumor suppressor gene KIF2B in HCC and provide a new insight in the mechanism.

课题前期利用高通量SNP芯片进行的关联研究，发现了位于KIF2B编码区的rs4561519（Arg→Gly错义突变）与肝癌存在直接相关性，肝癌组织中KIF2B的mRNA和蛋白表达水平显著低于癌旁正常肝组织。本项目进一步研究KIF2B突变及表达在肝癌发生的功能性作用及机制。通过分析肝癌、癌旁肝组织中KIF2B基因型及mRNA转录和蛋白表达水平，明确KIF2B基因型及表达与肝癌发生及预后的关系；对KIF2B启动子进行甲基化分析，探讨去甲基化药物干预的作用；构建KIF2B正常型和突变型表达质粒及KIF2B干扰质粒，研究KIF2B正常型或突变型基因表达抑制和过度表达后对肝癌细胞生长、增殖、凋亡、迁移及克隆形成的影响，通过裸鼠皮下成瘤实验观察KIF2B表达上调与下调的情况下对裸鼠皮下肿瘤生长的影响；分析KIF2B在肝癌细胞中的相互作用蛋白。本研究将阐明潜在抑癌基因KIF2B的功能，分析其作用机制。

肝癌的发生、预后是多因素、多机制的连续发展过程，由基因与环境之间的相互作用共同决定。本课题选择332例肝癌样本和328例对照样本，利用两阶段高通量SNP芯片进行全基因组扫描筛选与肝癌发生可能有关的SNP频率的分布差异，发现一个SNP位点（rs4561519）位于KIF2B基因的编码区，并造成错义突变（Arg→Gly）。SIFT、Polyphen和 SNPs3D上进行分析预测此突变会损害蛋白质功能。利用Realtime-PCR、免疫组织化学染色及Western blot检测发现KIF2B蛋白在癌旁肝组织中高表达，在肝癌组织中阴性或低表达。结合临床资料，采用Kaplan-Meier 方法比较显示KIF2B高水平表达能显著延长生存及减少复发，KIF2B甲基化水平与肝癌手术预后密切相关，但rs4561519 SNP位点的基因型与肝癌预后无关联。根据人类全长KIF2B基因的开放读码框序列设计引物，设计引物并以KIF2B基因ORF克隆为模板PCR扩增目的片段，将目的片段和突变片段分别构建表达慢病毒骨架载体，用构建的慢病毒载体和阴性载体分别转染293T细胞，包装病毒，细胞功能学检测发现HepG2和Huh7细胞过表达KIF2B基因后，可抑制细胞增殖，KIF2B突变后，抑制效果减弱；KIF2B基因过表达后，细胞周期阻滞在G1期，突变后，阻滞效果减弱；HepG2和Huh7细胞过表达KIF2B基因后，迁移能力减弱，KIF2B基因突变后，迁移能力有所恢复。HepG2和Huh7细胞KIF2B基因过表达可促进细胞凋亡，KIF2B突变后促凋亡能力减弱。裸鼠皮下成瘤实验发现HepG2各细胞株没有成瘤能力，而Huh7各细胞株成瘤效果良好。HuH7过表达KIF2B基因后，成瘤能力减弱，而KIF2B基因突变后，成瘤能力有所恢复。杂氮胞苷作用HepG2细胞后，目的基因KIF2B mRNA及目的蛋白表达均上调。进一步基因表达谱芯片结果显示KIF2B在Huh-7人肝癌细胞上调控RhoA信号、Regulation of Cellular Mechanics by Calpain Protease和Estrogen-mediated S-phase Entry等通路，并提示该基因在癌症、细胞死亡和病毒复制等疾病和功能中发挥作用。