肝细胞肝癌基于RUVBL1异常表达的预后列线图模型及其功能研究

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths in China with increasing annual incidence. Due to the lack of effective prognostic prediction and intervention strategies, about 90% of patients died from recurrence and metastasis with extremely poor prognosis. Nomograms are world-advanced, user-friendly graphical tools used for cancer prognosis. Because of their ability to reduce statistical predictive models into a single numerical estimate of the probability of an event (death or recurrence), they are tailored to the profile of an individual patient and easy to use during clinical encounters to inform clinical decision making. Our previous studies found that the mRNA and protein levels of RUVBL1 were increased in tumor cells, and significant translocation of RUVBL1 from nucleus to cytoplasm was observed. Its abundance was associated with higher tumor size, poorly differentiation and vascular invasion. Higher cytoplasmic RUVBL1 expression was an independent poor prognostic factor for relapse free and overall survival in HCC. It has a quite good prognostic performance for short-term HCC recurrence after surgery. Thus, we suspected that RUVBL1 might play important roles on invasion and metastasis of HCC cells. However, there are very limited reports about expression and function of RUVBL1 in HCC, and the molecular mechanisms underlying nucleocytoplasmic translocation, invasion and metastasis still remains unknown. In this study, the expression characteristics and the prognostic value of RUVBL1 in HCC will be investigated in four independent HCC cohorts using the exploration and validation mode. Meanwhile, the nomograms which combined the levels of RUVBL1 and prognosis-related clinicopathological parameters for predicting the probabilities of recurrence and survival after HCC resection will be constructed and evaluated in these four cohorts. Subsequently, the mechanisms of RUVBL1 promoting invasion and metastasis and nucleocytoplasmic translocation will be further explored in xenograft nude mice tumor models, experimental metastasis model via tail vein injection and in vitro HCC cells using the methods which combined transcriptome, interactome, post-translational modification-specific proteomics and functional studies. The results will provide useful biomarkers for predicting the prognosis and give a clue to potentially new target for intervention of HCC.

肝癌在我国高发，由于缺乏预后预测体系且无法有效干预，90％以上的患者死于复发和转移。申请人前期工作发现RUVBL1在肝癌中高表达，且存在核浆转位现象，其丰度与肿瘤大小、分化程度、血管侵犯、短期复发和不良预后等相关，因此推测胞浆中的RUVBL1参与肝癌侵袭转移过程。而目前关于其在肝癌中的表达和功能研究很少。本研究拟利用四个肝癌队列，系统探讨RUVBL1在肝细胞癌变不同阶段中的表达变化及其预后判断价值；通过国际先进的列线图模型方法，建立并验证适于中国人群且结合RUVBL1和临床病理参数的，能预测术后复发和生存概率的列线图模型；随后通过转录组和相互作用/翻译后修饰蛋白质组、体内和体外功能实验相结合的策略，明确RUVBL1核浆转位，及其促进肝癌侵袭转移的具体机制。研究结果将建立适宜临床使用且准确度更高的肝癌复发和生存概率模型，明确RUVBL1在肝癌侵袭转移中的作用，此外为寻找新的干预靶点奠定基础。

申请人基于TCGA RNA测序数据发现，RUVBL1 mRNA在肝癌组织中上调，并与不良分化、血管侵犯和短的总生存期及无复发生存期相关。免疫组化显示，RUVBL1蛋白在正常肝和癌旁正常肝组织主要定位于细胞核，而肿瘤组织中核定位的RUVBL1明显减少，胞浆定位显著增加，存在明显的核浆转位现象。无论是在完全正常肝-肝硬化-肝癌的三阶段进程中，还是在完全正常肝-肝硬化-高分化肝癌-中分化肝癌-低分化肝癌的五阶段演进过程中，RUVBL1的核浆转位均呈梯度增加趋势。胞浆中的RUVBL1水平是预测HCC患者无复发生存时间的独立危险因素。由此建立了包括胞浆RUVBL1染色强度在内的，由6个临床病理指标组成的列线图模型，对术后复发概率和总生存概率预测的一致性指数分别为0.7和0.73，显著优于BCLC和TNM临床分期系统。RUVBL1是经典的核蛋白，但上述发现提示，胞浆中异位表达的RUVBL1才是驱动肝癌进展的主要因素。随后的功能研究发现，RUVBL1可以显著增加肝癌细胞的增殖、长期存活、运动和侵袭能力。而胞浆中的RUVBL1可以通过与CTTN等蛋白形成功能复合物，促进细胞的运动和侵袭。此外，我们也发现RUVBL1的伴侣蛋白RUVBL2同样在肝癌中高表达，但其表达特征与RUVBL1并不一致。肿瘤组织中细胞核和细胞浆定位的RUVBL2蛋白均显著高于癌旁，两者分别是预测患者总生存时间和无复发生存时间的独立危险因素。RUVBL2可通过HSP90-CDC37复合物活化下游AKT和ERK通路，进而促进肝癌细胞的增殖、克隆形成、运动和侵袭。因此RUVBL1和RUVBL2在肝癌发生发展中可能发挥不同的功能，两者均可作为有前景的HCC预后标志物以及潜在治疗靶标。课题执行期间发表标注基金号的SCI文章10篇，其中第一标注8篇；核心期刊5篇；申请专利2项，授权专利2项。培养研究生5人。