RNA过编辑现象与肝癌发生发展的关系研究

Liver cancer, hepatic cellular carcinoma (HCC), is one of the common cancers world-wide, and the mechanisms of hepatocellular oncogenesis are unclear. With the development of high throughout sequencing technology, we can find many novel genetic events involving into oncogenesis. We have integratedly analyzed the editome of HCC using whole genome and transcriptome sequencing data. And then found 68100 RDD sites (RNA and DNA Diference) in 2 paired of HCC s and 2 HCC cell lines. According to a strict criterion, we in total identified 3623 RDD sites had significantly differential expression level in two pairs of HCCs and non-HCCs. Including 85 non-synonymous sites involving into 75 genes. Of which 7 genes with tumor-specific RDD events displayed high frequency overediting in two HCCs. And then sequenom MassARRAY technology was used to genotype validation of the 7 RDD sites in DNA and cDNA samples of the two pairs of HCCs and non-HCCs we found that 5 out of 7 genes were confirmed overediting in HCCs by using sequenom MassARRAY technology. Of which we found that BLCAP gene had a higher frequency of nonsynonymous A→I transcript editing, leading to a Ser→Gly amino acid substitution in the two paired of HCCs and non-HCCs. To confirm the result, we investigated BLCAP editing in 179 matched primary HCC and nontumor liver tissues using sequenomMassARRAY. Our result showed that BLCAP gene expression with RDD events was significantly higher in HCCs (P<0.0001), and about 40% of the primary HCC specimens had BLCAP gene overediting. Notably, clinicopathological analyses demonstrated that BLCAP overediting in tumors was significantly correlated with worse prognoses (P=0.0006). Significantly, BLCAP overediting and AFP did not completely overlap in the studied HCC specimens. BLCAP overediting was evident in 42% of 50 HCC specimens with AFP negative, suggesting that the potential of BLCAP gene overediting may be serviced as a novel biomarker for HCC pathologenesis. The objective of this project is first to study the relationship between BLCAP overediting and hepatocarcinogenesis in a large of clinical samples. The second part will involve testing the hypothesis that BLCAP overediting will play a functional and causal role in the hepatocellular oncogenesis. This will be done by a systematic overexpression of BLCAP_WT and BLCAP_RDD genes or by a systematic siRNA knockdown of BLCAP gene into HCC cell lines, then observe the phenotyping changes and identify the functional pathways causally involved in hepatic cancer. The third part will involve testing of the molecular mechanisms involving into BLCAP overediting leading to hepatic cancer. In summary, the project's aim is to chart the editome of HCC, identify the critical players like BLCAP and so on to establish a basis for profound understanding of hepatocellular oncogenesis, and subsequent lay the foundation for developing the potential biomarker for diagnosis and therapeutics of liver cancer through this study.

肝癌是恶性肿瘤之一，预后很差，机制并不十分清楚。我们前期利用高通量测序技术整合分析肝癌样本全基因组和全转录组数据，发现肝癌细胞中有大量的基因存在RNA编辑现象，经过严格的生物信息学筛选，发现BLCAP基因在肝癌组织有过编辑现象。大样本验证结果显示40.1%的肝癌组织中发生BLCAP过编辑，BLCAP过编辑与肝癌患者的较短生存期有密切的关系，提示预后不良；同时BLCAP在22.37%AFP阴性肝癌组织中过编辑。前期结果显示BLCAP过编辑可能在肝癌的发生发展过程中发挥着重要作用。为了更深入地研究BLCAP过编辑现象与肝癌的关系，本研究拟在细胞和动物水平深入研究（1）BLCAP 过编辑与肝癌发病的关系；（2）BLCAP 过编辑对肝癌细胞行为学特征的影响及其相关的信号通路；（3）BLCAP 过编辑发生的分子机制研究，为阐明肝癌的发病机制提供新的理论依据，为开发肝癌的临床诊疗技术提供新的思路。

肝癌是严重影响人的健康的恶性肿瘤之一。目前，对于肝癌的早期诊断和治疗虽然有了一定认识，但肝癌预后仍然很差，特别是对其发病机制的了解，更是知之甚少。为了更好的在分子层面探究肝癌的治病机理，我们利用高通量测序技术分析整合了两对肝癌组织样本的全基因组和转录组数据。发现BLCAP基因在肝癌中是一个新的有过编辑现象的基因，并发现在40.1%的肝癌组织发生BLCAP 过编辑。我们利用RNA 干扰和基因转染技术评估了BLCAP RNA过编辑在肿瘤进展中的作用。结果显示，与野生型的BLCAP 基因相比，RNA 过编辑的BLCAP基因可能促进了细胞的增值（包括细胞生长，克隆形成及体内的成瘤能力），这种作用是通过增强AKT, mTOR以及MDM2的磷酸化，或者抑制TP53的磷酸化来实现的。我们目前的实验结果表明BLCAP 基因的过编辑可能是一个新的潜在的驱动基因，通过激活AKT/mTOR信号通路来促进肝癌的发展。