c-jun在sorafenib诱导肝细胞癌自噬过程中的作用及机制的研究

Sorafenib, currently, is the only effective targeted drug in the patients with advanced hepatocellular carcinoma(HCC). However, sorafenib may cause resistance in many patients and it is imminent to investigate the involved mechanisms. Recently we demonstrated an increased expression of c-Jun after treated with sorafenib in HCC cells, which was assosiated with the inhibitory effect of sorafenib on HCC cells, and we also showed a high expression of p-c-Jun was associated with poor prognosis in sorafenib-treated advanced HCC patients. These results showed a possible c-Jun mediated resistance to sorafenib in HCC cells. The involved mechanisms seem to be that the increased c-Jun expression induced autophagy of HCC cells and thus cause resistance, through the mechanism of endoplasmic reticulum (ER) stress, BCL-2 and the downstream signalling pathway of c-Jun. We have confirmed the autophagy phenomenon induced by sorafenib in HCC cells in our preliminary study. In the present study, we intend to use the methods including siRNA, c-Jun overexpression vector in cells, luciferase reporter gene method and chromatin immunoprecipitation assay(CHIP), to investigate a new mechanism causing resistance of HCC cells to sorafenib by the increasing expression of c-Jun, to confirm the autophagy induced by c-Jun and explore the possible regulatory mechanisms. The present study would shed a new light on the personal therapy of sorafenib and provide a potential new molcecular target for the disease of HCC.

Sorafenib是治疗晚期肝癌（HCC）有效的唯一靶向药物，但临床发现部分患者敏感性差，机制需要进一步研究。我们前期研究发现sorafenib作用肝癌细胞后c-Jun表达上调，c-Jun表达和sorafenib抑制HCC的疗效密切相关，高表达p-c-Jun分子的HCC患者预后较差，即c-Jun可能介导了HCC对sorafneib的耐受。分析发现c-Jun可能通过诱导自噬实现对HCC细胞sorafenib耐受；内质网反应、BCL-2分子和c-Jun的下游信号机制参与此过程。我们的预实验已证明sorafenib诱导HCC细胞自噬，本项目拟采用siRNA，真核重组表达载体，荧光素酶报告基因法，CHIP等方法，旨在揭示c-Jun在HCC中诱导自噬介导sorafenib耐受的新机制，阐明具体通路和机制，为sorafenib“个体化”治疗HCC提供新的思路和分子靶点。

Sorafenib是目前唯一显著延长晚期肝癌（HCC，Hepatocellular carcinoma）患者生存的分子靶向药物，但临床发现其疾病控制率在43%左右，意味并非所有的HCC患者能从中获益。HCC可能通过自噬机制对sorafenib产生药物耐受。申请者前期发现c-Jun分子与sorafenib抑制HCC疗效密切相关，推测其可能通过自噬机制参与耐药机制。.申请者获得资助后，初步研究提示c-Jun在sorafenib耐药机制中的作用，不符合课题所设想的预期研究目标。经过文献分析，推测可能和自噬存在多种调控机制有关，后续结合目前研究的热点领域，围绕“circRNA分子、sorafenib、自噬”三者关系进行进一步的研究。实验方案调整后，申请者构建了针对sorafenib的Huh-7肝癌耐药细胞株，通过多种方法验证耐药细胞株构建成功；对Huh-7与Huh-7R细胞株进行circRNA测序并进行比较，结合文献，选择自噬机制密切相关的信号通路，应用生物信息学分析，筛选得出hsa_circ_0002404，hsa_circ_0001495这两个候选circRNA分子； HCC组织肿瘤及癌旁标本中的验证结果表明hsa_circ_0002404，hsa_circ_0001495这两个circRNA分子可能与HCC的发生发展亦有密切关系；进一步构建hsa_circ_0002404慢病毒过表达载体，在Huh-7细胞中瞬时转染，qPCR检测过表达成功，为后续进行hsa_circ_0002404环状分子功能实验作准备。.课题经费目前执行超过70%，后续拟继续对筛选出来的候选分子进行临床验证其与sorafenib耐药的深入联系，进行功能验证，并探讨是否存在circRNA-miRNA或circRNA-miRNA-mRNA机制参与“自噬导致HCC对sorafenib耐药机制”。课题的最终完成，将为肝癌的分子靶向治疗提供更广阔的思路与科学依据。