肝癌转移时胞嘧啶核苷脱氨酶（AID）对AXL基因去甲基化的调控

Activation-induced cytidine deaminase（AID）belonges to the APOBEC family, which induce DNA or RNA cytidine （C）-to- uracil（U）mutation. I focus on AID research for a long time, and have published numerous research articles in PNAS, PLoS Pathogens, etc. AID could induce cancer formation and metastasis, which has been reported, but the mechanism remains to be clarified. Our preliminary results showed that AID could induce EMT level. AXL is the receptor tyrosine kinase (RTKs), which is important in tumor metastasis. Demethylation of AXL gene could induce tumor metastasis, which has been reported. It is well known that AID could induce DNA demethylation. Other group showed that, high expression level of AXL is the reason of tumor drug resistance associated with EGFR family. Based on these works,we also hypothesize that tumor drug associated with EGFR family could increase AID expression，which could induce AXL activation through demethylating AXL gene,then high level of AXL induces EMT upregulation, resulting in hepatocellular carcinoma metastasis.To confirm these,our preliminary results showed that 1. The expression level of AID and AXL from hepatocellular carcinoma cells are more than normal liver cells. 2. AID could demethylate AXL gene, and upregulate AXL expression level. 3. AID could induce EMT level through AXL. 4. High AXL expression level in tumor drug resistance is due to AID. Next we want to clarify and confirm that high level of AXL expression inducing EMT and tumor drug resistance demethylated by AID. Our project will provide a new theoretical direction for clinical treatment or pharmaceutical.

AID是胞苷脱氨酶家族的一员，具有DNA超突变及抗体类别转换的功能。已证明AID的异常表达会促进肿瘤形成、转移，但机制不明确。AXL在癌症的转移中起重要作用。有文献报道，AXL导致的EMT的高表达与EGFR家族抗肿瘤药物耐药性的形成有关。我们前期的实验数据表明：1. EGFR家族抗肿瘤药物可以提高AID的在肝癌细胞中的表达；2. AID的异常表达可以通过上调AXL的表达显著上调EMT。进一步的实验表明AID可以下调AXL基因的DNA甲基化水平。因此我们提出科学假说：在肝癌转移中，靶向EGFR家族抗肿瘤药物可以诱导肝癌组织中AID的异常表达，介导AXL基因的去甲基化，引起AXL的高表达，导致肝癌的转移，产生耐药性。本研究旨在通过甲基化检测、CRISPER Cas9、免疫组化等实验方法，揭示AID调控AXL在肝癌转移中的新机制，同时为确立AID作为癌症治疗的新靶点提供更充分的的科学依据。

背景：近年来，很多报道显示1.胞嘧啶脱氨酶家族中的AID具有超突变、类别转换的功能，并且与肿瘤的发生发展密切相关；2.AXL的异常表达能引起上皮-间质转化（EMT）；3.EGFR药物上调AXL是引起的肿瘤耐药性形成的原因之一，但是上述机制都不完善。内容：基于申请人既往在国际上首次报道AID可以抑制HBV的复制等相关研究（申请人导师为AID发现人），本项目讨论了AID诱导AXL异常表达能引起EMT。更重要的是AID促使AXL异常表达在EGFR引起的耐药性过程中起着重要的作用。结果和数据：1.AID与AXL在肝癌细胞中呈现高表达。2.AID在肝癌细胞中能促进AXL的表达，但在其他癌症细胞中AXL没有明显的差异。3.AID通过提高AXL促进EMT。首先，过表达AID、AXL均可以促进EMT的表达。当敲除AID、AXL之后，EMT则被抑制。其次当敲除AXL之后过表达AID，EMT则不会被影响。4.EGFR类药物可以同时提高AID、AXL的表达，更重要的是，敲除AID之后，加入EGFR类药物也不会影响AXL的表达水平。5.对过表达AID的肝癌细胞进行全RNA测序，找出与AID相关的mRNA、lncRNA、miRNA等，丰富AID与肝癌的机制。科学意义：AID可能是肝癌发生及发展的新的生物学标志物，并为肿瘤的发生发展、药物筛选提供了一种新的思路和科学依据。