HBX蛋白通过LASP-1与vimentin相互作用促进肝癌细胞EMT的机制研究

The hepatitis B virus (HBV) X protein (HBX) plays a key role on epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC). We found that HBX could interact with vimentin, a molecular marker of EMT, to inhibit the ubiquitination of vimentin, enhance the stability of vimentin and promote it expression. Skeleton protein LASP-1 is found to be involved in the interaction of HBX and vimentin and participate in EMT of HCC. Furthermore, inhibiting the expression of LASP-1 could weaken the interaction between HBX and vimentin and decrease the expression of vimentin in HCC cells. These results suggest that HBX could interact with vimentin via LASP-1, and the ubiquitination and stability of vimentin mediated by HBX is dependent on LASP-1, to promote the EMT of HCC cells. The project intends to construct the expression vectors and associated mutation vectors of HBX, LASP-1 and vimentin, to find the key binding sites of each proteins with co-immunoprecipitation and other assays. To further explore the mechanism of interaction of HBX with vimentin via LASP-1 to inhibit the ubiquitination of vimentin, increase the stability of vimentin and facilitate the EMT of HCC cells. This study will provide useful ideas for targeting EMT in HBV-related HCC.

乙肝病毒（HBV）X蛋白（HBX）对肝癌细胞的上皮间质转化（EMT）起关键作用。申请者发现HBX在致肝癌细胞EMT的过程中能够与EMT分子标志vimentin相互作用，导致vimentin泛素化降低、稳定性增强、表达量增加。并且骨架蛋白LASP-1可与HBX、vimentin相互作用参与肝癌细胞的EMT；抑制LASP-1的表达可显著减弱HBX与vimentin的相互作用及vimentin的表达，提示HBX可通过LASP-1与vimentin相互作用，介导vimentin的泛素化和稳定性，促进肝癌细胞的EMT。本项目拟构建三种基因的表达载体及突变载体，采用免疫共沉淀等实验，明确HBX、LASP-1和vimentin相互作用的关键作用位点；并深入探讨HBX通过LASP-1抑制vimentin泛素化、增强其稳定性，促进肝癌细胞的EMT的分子机制，为开发靶向EMT的HBV相关肝癌治疗提供有益思路。

乙型肝炎病毒（hepatitis B virus, HBV）的慢性感染是导致肝癌发生发展的重要原因。目前的研究表明，HBV X蛋白（HBX）与HBV相关性肝癌的上皮间质转化（Epithelial mesenchymal transition, EMT）密切相关。vimentin蛋白是一种EMT相关的分子标志。然而vimentin在HBX介导的肝癌发生发展中的作用及相关的分子机制尚不明确。在本研究中，通过western blot和免疫组化等方法，我们发现，相对于对照细胞和癌旁组织，vimentin蛋白在HBX阳性肝癌细胞和HBV相关的肝癌组织中的表达显著增加；并且在HBV相关的肝癌组织中，vimentin蛋白与HBX的表达显著相关。分子功能上的研究表明，vimentin参与HBX介导的肝癌细胞的EMT、增殖和迁移。对其进一步的研究发现，在细胞水平，HBX可通过LASP1蛋白促进vimentin蛋白的表达，介导肝癌细胞的EMT。在组织水平，LASP1蛋白与vimentin蛋白在肝癌组织中的表达显著相关。相关的分子机制的研究表明，在肝癌细胞中，HBX可以直接与vimentin和LASP1蛋白相互作用，并且依赖于LASP1，HBX能够通过保护vimentin蛋白免受泛素蛋白酶体途径介导的蛋白质降解，从而提高vimentin蛋白的稳定性，促进vimentin蛋白的表达。综上所述，依赖于LASP1，vimentin蛋白在HBX介导的肝癌细胞的EMT及相关细胞功能异常中发挥重要的作用，vimentin及其调控蛋白LASP1可能成为HBV相关肝癌的治疗的潜在分子靶点。