肝癌中ATAD2基因两种异构体的功能研究及其诱导型转基因斑马鱼肝癌模型的建立

To find more oncogenes systematically which may have variations on transriptional level in hepatocellular carcinoma tisses compared with the paired adjacent tissues and generate new animal model for HCC,we set up a research based on analysis of RNA-seq data of 10 paired HBV-affected hepatocellular carcinoma tissues.We specially focused on the screening of new alternative splicing events and found a new isoform of ATAD2 that is expessed dramatically higher in HCC tissues than in the adjacent tissues. The experimental strategy utilized in the validation of the function of ATAD2 in HCC is to finish the following in successive steps. We first verify the expression of two isoforms in paired HCC and adjacent tisses.The transcripts of two isoforms of ATAD2 are obtained from the cDNA of the HCC tisses by PCR directly, and are utilized in comparison in overexpression or inhibition in different HCC cell lines as well as normal liver cell lines in vitro. The characteristics of the transformed cell lines will be examined, such as the ability to proliferate, cell cycle and cell apoptosis to check whether ATAD2 is oncogenetic in HCC and the exact mechanism behind each isoform. Then the two isoforms are tested on nude mice for tumor formation in vivo. Finally, we will generate a mifepristone-induced transgenic zebrafish model with the two isforms of ATAD2 specifically overexpressed in liver seperately or collectively, which may develop carcinoma in the liver after mifepristone inducement. The transgenic zebrafish model of ATAD2 can be further applied in medicine screening in HCC. Our research will clarify the mechanism underlying the alternative splicing of ATAD2 gene in HCC and shed light on new target genes in HCC diagnosis and therapy.

为更加系统全面的筛选转录组水平变化的潜在致癌基因并构建相应转基因动物模型，本研究拟以前期工作中10对HBV感染肝癌组织样本的转录组测序数据为基础，重点关注转录组水平发生的选择性拼接的改变，发现ATAD2基因在肝癌组织中比癌旁组织显著高表达一种新的异构体。完成差异表达的组织验证后，从肝癌组织中通过PCR直接获取两种异构体的转录本,完成体外细胞功能实验和裸鼠成瘤实验验证；在此基础上，运用米非司酮诱导外源基因表达的方法建立ATAD2两种异构体分别或共同在肝脏中特异诱导表达的转基因斑马鱼肝癌模型，进行两种异构体致癌机制的体内功能验证及对比分析。本研究将解释选择性拼接对ATAD2在肝癌中功能的影响，阐明ATAD2基因在肝癌发生发展中的作用机制。ATAD2的转基因斑马鱼肝癌模型将为高通量筛选治疗肝癌的小分子药物提供平台，预期研究成果为肝癌靶向治疗提供重要的实验依据。

该研究以前期工作中10对肝癌组织样本的转录组测序数据分析为基础，重点关注ATAD2基因的在肝癌中的表达水平差异以及新的转录异构体的出现。项目计划完成差异表达的组织验证后，进行体外细胞功能及裸鼠成瘤实验，并在此基础上运用米非司酮诱导外源基因表达的方法，建立ATAD2在肝脏中特异诱导表达的转基因斑马鱼模型。我们的研究结果最终表明，ATAD2在RNA及蛋白水平上均在肝癌组织中高表达，但未在大数量样本病例中证实ATAD2的新异构体形式，证明该异构体并非普遍现象。体外的功能实验证明ATAD2的RNA水平改变会影响细胞的凋亡及细胞周期，并影响细胞的迁徙能力。ATAD2在肝脏中特异诱导表达的双转基因鱼中的功能还有待最终阐明。本研究主要提示了ATAD2基因在肝癌发生发展中可能的作用机制。同期筛选出的肝癌差异表达基因GTSE1在肝癌及结直肠癌中的临床相关性研究也获得了初步结果。