GTPBP4通过Akt/GSK-3β/p53途径影响肝细胞癌恶性表型的机制研究

So far，the research on the influence of GTP binding protein 4（GTPBP4） to the oncogenic phenotypes of tumor is little. Our previous studies found that GTPBP4 highly expressed in clinical speciments of hepatocellular carcinoma, the malignant phenotype of hepatocellular carcinoma cells was down-regulated after GTPBP4 silenced，and overexpression of GTPBP4 may suppress the activation of p53 in hepatocellular carcinoma cells. However, the report pointed out that high expression of GTPBP4 led to p53 inactivation, which was independed on the ubiquitination pathway. It is well known that phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) can inhibit the activation of p53 by inactivation of the glycogen synthase kinase 3β(GSK-3β) phosphorylation as well as acetylation of p53. In other hand, GTPBP4 can activate the PI3K. Therefore, we hypothesize that GTPBP4 can lead to inactivation of p53 and promote the oncogenic phenotypes of hepatocellular carcinoma through the above mechanisms. This project intends to construct hepatocellular carcinoma cell lines with GTPBP4 lower expression and re-expression, observe the influence of GTPBP4 on hepatocellular carcinoma cell proliferation, apoptosis, and the aggressivity at cellular and molecular levels both in vivo and in vitro, prove the function of GTPBP4 to regulate the acetylation of p53 at the level of protein,take advantage of specific inhibitors and dominant negative mutants to intervene the Akt/GSK-3β/p53 pathway, and evaluate the mediating role of the Akt/GSK-3β/p53 pathway in the above functions that GTPBP4 exerts. Providing a novel targets for gene therapy of hepatocellular carcinoma.

关于GTPBP4基因对肿瘤恶性表型影响的研究甚少，我们前期研究发现其在肝癌组织中高表达，其表达沉默后肝癌细胞株恶性表型下调，此外，研究结果还提示，GTPBP4高表达可阻遏p53激活。然而，有报道指出，GTPBP4是不通过泛素化途径阻遏p53激活的。已知PI3K/Akt可使GSK-3β磷酸化失活从而抑制p53乙酰化以阻遏其激活，而GTPBP4可激活PI3K。因此我们假设，GTPBP4可经上述机制阻遏p53激活进而上调肝细胞癌的恶性表型。本项目拟构建GTPBP4低表达和表达回复肝癌细胞株；在细胞和分子水平观察GTPBP4对肝癌细胞增殖、凋亡及侵袭的影响，并在多株肝癌细胞中和动物整体水平上进行验证；从蛋白水平探明GTPBP4对p53乙酰化的调控作用；运用特异性抑制剂和显性负突变体对Akt/GSK-3β/p53途径进行干预以评价其在GTPBP4发挥以上功能中的介导作用，旨为肝癌基因治疗提供新靶点。

GTPBP4是GTP连接蛋白家族成员之一, 已知，GTPBP4在乳腺癌、结肠癌等肿瘤的发生发展中可起到推进作用，然而仅为初步探索，并未对其中的具体机制进行阐述，关于GTPBP4在肝细胞癌（HCC）发生发展中作用及其机制的研究仍鲜有报道。本项目旨在揭示GTPBP4促进肝癌进展的调控机制。本研究按步骤的完成年度研究计划，我们利用生物信息学及分子生物学技术，发现GTPBP4基因下调后改变了调控细胞周期的关键基因如CCND1，CCND2，CDK6、MDM2等的表达，提示GTPBP4可对细胞周期进行调控。另外我们发现，miR-101-3p/WEE1轴可下调lncRNA NEAT1_2对肝细胞癌细胞的放射增敏作用，WEE1属于丝/苏氨酸蛋白激酶家族,其可抑制细胞进行有丝分裂,调节细胞周期进展，WEE1可与PI3K/Akt协同推进肿瘤的进展，因此我们认为，GTPBP4可能通过上调WEE1的表达从而影响Akt/GSK-3β进而调控包括p53在内的细胞周期关键基因表达。此外，我们在GTPBP4的上游发现了一个名为LINC00346的长链非编码RNA，我们发现LINC00346可通过调控miR-873-5p/β-连环蛋白轴从而促进HCC的进展，接下来，我们发现在肝细胞癌细胞中存在一个涉及LINC00346、β-连环蛋白、MYC的正反馈环路发挥促进HCC进展的作用。最后我们还发现FGD5-AS1可通过调miR-873-5p/GTPBP4轴从而促进HCC细胞的增殖、迁移和侵袭，并抑制其凋亡。综上所述，GTPBP4可调控细胞周期促进肝癌的进展，并且其受到上游长链非编码RNA的靶向调控。