新型致癌基因AEG-1调控NF1/AKR1C2基因表达及上皮间质转化(EMT)促肝癌转移的机制研究

Astrocyte elevated gene 1 (AEG-1), as a novel oncogene, has recently been reported that it plays a critical role in epithelial-mesenchymal transition (EMT) and metastasis of hepatocellular carcinoma. However, its precise role is still unknown in hepatocellular carcinoma. Our previous studies found that the PI3K-Akt expression levels, morphology and invasion of hepatocellular carcinoma cells were changed through inhibition of AEG-1 expression. Using RNA-seq-based gene expression profiling, we identified two AEG-1 target genes (NF1 and AKR1C2 genes) after AEG-1 gene silencing. The results showed that their expressions were correlated with metastasis of hepatocellular carcinoma. By experiments in vitro and in vivo, we will further investigate whether and how AEG-1 pathway regulates NF1 and AKR1C2 genes, and induces epithelial-mesenchymal transition (EMT) and metastasis of hepatocellular carcinoma. Using co-immunoprecipitaion, immunofluorescence and luciferase reporter system technology, we further explore the AEG-1, NF1 and AKR1C2 genes involved in regulating the process of hepatocellular carcinoma epithelial-mesenchymal transition (EMT) and metastasis; determine how the interaction between them; detect whether PI3K-Akt as an essential factors of AEG-1 target genes (NF1 and AKR1C2 genes) activation. These findings will provide new insights into mechanisms how AEG-1 pathway promotes hepatocellular carcinoma metastasis, and will prove its potential therapeutic target value for hepatocellular carcinoma patients.

AEG-1是近几年发现的新型癌基因，已证实其可通过调控上皮间质转化(EMT)进而促进肝癌细胞的转移，但其发挥作用的确切机制尚不清楚。我们前期研究发现：抑制AEG-1基因表达，肝细胞癌的侵袭转移能力和PI3K/Akt表达水平发生变化；应用二代测序技术筛选发现沉默AEG-1基因后NF1和AKR1C2基因差异表达最为显著，经鉴定二者与AEG-1表达及肝癌转移密切相关。因此，我们假设:AEG-1调控NF1/AKR1C2基因表达及EMT促肝癌侵袭和转移。本项目拟从体内、外两个层面，用荧光素酶报告基因、免疫共沉淀、动物肿瘤荧光成像、免疫组化等方法，基于PI3K-Akt信号传导通路研究AEG-1调控NF1/AKR1C2基因表达及上皮间质转化促肝癌侵袭转移的新机制。本研究探索AEG-1基因的调控方式，为肝癌复发及转移高危人群的临床诊断提供一个新的指标，并为基于EMT机制的肝癌消融提供了一个潜在的新靶点。

肝细胞癌病人手术后复发和转移率高，预后差。但其发生机制因其具有不同的遗传异质性，一直都没有研究清楚，也成为我国开展肝细胞的精准医疗亟待解决的问题。因.此申请者在本课题中：1) 明确了AKR1C2 和 NF1 具有促进肝癌细胞上皮间质转化（EMT）和侵袭转移能力的作用。2) 首次阐释了在肝细胞癌上皮间质转化（EMT）和转移过程中 AEG-1 过转录抑制PIK3R1来激活PI3K-Akt通路，从而间接对 AKR1C2 和NF1 表达的调控方式及其作用关系。3)进一步 明确了临床标本 AEG-1、AKR1C2 和 NF1 三者表达水平与上皮间质转化标记蛋白的相关性，证实了AEG-1/PI3K-Akt/NF1- AKR1C2的调控通路的存在性。通过以上研究，进一步完善以AEG-1为中心的信号网络，在分子水平上阐明AEG-1基因促进肝癌上皮间质转化和转移的机制，为肝癌的临床治疗寻找新的分子靶标。为肝癌复发及转移高危人群的临床诊断提供新的指标， 并为基于 EMT 机制的抗肝癌治疗提供了潜在的新靶点。