核糖体自身翻译起始阻滞在肝癌发病中的作用机制研究

Hepatocellular cancr, with a fast-growing morbidity every year, has ranked the third place in motality of all cancer types. Considerable efforts have been put to investigate hepatic tumorigenesis, which revealed viral or bacterial induced liver inflammation, epithelial-mesenchymal transition, hypoxia and other cell signalling pathways to be the key affecting factors. However, none of the previous researches have studied changes in protein translation and its regulation during the hepatic tumorigenesis process. To solve this problem, we set up a method called Ribosome-protected fragments deep-sequencing (Ribo-seq), which is able to measure the footprints and numbers of ribosome on mRNA at necleotide resolution. Employing this method, we have shown that in HepG2 cells, a hepatic cancer cell line, the polysome-binded mRNA numbers as well as the ribosome occupancy are higher than in a normal hepatic cell line L02 cells. In addition, we also found that ribosomal translational initiation pausing (iPause), which may be regulated by Terminal oligopyrimidine (TOP) sequence, is lower in HepG2 cells than in LO2 cells. Taken the previous results together, Ribo-seq is an effective approach to study the protein translational changes in hepatic tumorigenesis. Therefore, we propose to fverify the difference of the ribosomal translational initiation pausing between human hepatic cancer cells and normal tissue in details, and to further determine whether the TOP sequence is the key regulator of the ribosomal translational initiation pausing, using the Ribo-seq technique. This research will be the first one in the world to study the role of mTOR signaling pathway in the hepatic tumorigenesis process.

肝癌全球发病率在每年增长，其死亡率占癌症死亡率的第三位。已报道肝癌的发生受到病毒或细菌等引起的炎症反应、上皮-间质转换、低氧及诸多信号分子通路的影响。而肝癌发生过程中肝细胞内的蛋白质自身翻译的改变及调控尚未见报道。我们前期建立了核糖体保护片断深度测序技术，可以在核苷酸水平定位核糖体在信使RNA的位置。前期研究结果显示肝癌细胞系HepG2多核糖体结合的信使RNA数量及信使RNA的核糖体结合率明显高于肝脏正常细胞系LO2；核糖体蛋白自身翻译起始阻滞在肝癌细胞明显低于肝正常细胞；TOP序列有可能调控核糖体自身翻译阻滞。本项目拟继续采用核糖体保护片断深度测序技术， 进一步验证核糖体蛋白自身翻译起始阻滞在肝脏在体肿瘤及肝脏正常组织的差异，进一步证明TOP序列对糖体蛋白自身翻译起始阻滞的调控，并证明mTOR信号通路对这一过程的作用。

机体内很多器官可以承载病毒地复制和生存，但是机制却不是很清楚。我们的研究发现， 在SENP3的介导下，肝脏细胞可以通过提高宿主细胞自身蛋白质翻译对抗乙肝病毒的感染。利用嘌呤霉素标记技术和核糖体保护片段高通量测序技术， 我们的研究证明HBX 蛋白的表达诱导肝脏细胞内SENP3的表达降低，从而提高宿主细胞蛋白质整体表达增加。我们的研究进一步显示蛋白IQGAP2是SENP3介导宿主细胞翻译增加的重要底物， 敲除IQGAP2后， 磷酸化的AKT表达增加，进而提高宿主细胞蛋白质的整体翻译，同时抑制HBV基因的表达。SENP3-IQGAP2 去SUMO化的轴线显示了宿主细胞对病毒感染的适应性反应， 并且通过提高宿主细胞的翻译增加细胞的生存。