环状RNA hsa_circ_0005785作为新的肝癌生物标志物及其作用机制研究

Circular RNA plays an important role in the occurrence and development of the malignancies. Our previous studies have found that hsa_circ_0005785 is highly expressed in hepatocellular carcinoma (HCC) and has a positive correlation with the expression of a proliferation-inducing ligand (APRIL). Both of them can promote HCC growth and metastasis. According to the results of preliminary experiments, we speculate that hsa_circ_0005785 can competitively combine with miR-383 to facilitate APRIL expression in HCC. On the other hand, HCC cells can recruit tumor-associated neutrophils (TANs) which release APRIL through hsa_circ_0005785 binding chemotactic factor CXCL5. Furthermore, APRIL, derived from autocrine and paracrine, activates the NF-kappa B signaling pathway and enhances CXCL5 gene transcription. Both of them form a positive feedback regulatory loop to accelerate the progress of HCC. Accordingly, the aim of this project is to explore the molecular mechanisms of hsa_circ_0005785 acting as a “miRNA sponge” and participating in the "neutrophils-tumor interaction dialogue" to promote the malignant progress of HCC. In addition, a qPCR method to detect plasma hsa_circ_0005785 will be established and its methodology will also be evaluated in order to explore its clinical application values as a promising biomarker for HCC. Thus, our study might provide a novel idea and target for study of the pathogenesis, as well as clinical diagnosis and treatment of HCC.

环状RNA在肿瘤发生发展过程中起重要作用，我们前期发现hsa_circ_0005785在肝细胞肝癌（HCC）中高表达，与增殖诱导配体（APRIL）表达呈正相关，促进HCC生长转移。根据预实验结果，推测hsa_circ_0005785可通过竞争结合miR-383，促进HCC细胞表达分泌APRIL；还可通过绑定趋化因子CXCL5，增强其趋化能力，促使肿瘤相关中性粒细胞浸润并释放APRIL。自分泌和旁分泌的APRIL进一步激活NF-κB通路，增强CXCL5基因转录，二者形成正反馈环路，加速HCC进展。本项目旨在解析hsa_circ_0005785通过充当“miRNA海绵”及参与“中性粒-肿瘤交互对话”促进HCC恶性进展的分子机制。同时，建立并评价qPCR法检测血浆hsa_circ_0005785含量，探讨其作为新的HCC生物标志物的临床应用价值。本研究有望为HCC基础与临床研究提供新思路和靶标。

肝细胞肝癌（HCC）是我国最常见的恶性肿瘤之一，其确切致病机理仍未完全阐明。环状 RNA（circRNA）是一类呈闭合环状结构的非编码 RNA分子，异常表达的 circRNA在肿瘤发生发展过程中起重要作用。本课题组利用circRNA芯片筛选，并经qPCR证实hsa_circ_0005785在HCC癌组织中高表达，且与HCC患者TNM分期及预后相关。经RNase R耐酶试验及Sanger测序分析显示hsa_circ_0005785具有完整的环状结构。hsa_circ_0005785在HCC细胞株中明显升高，构建针对hsa_circ_0005785干扰载体转染HCC株SK-Hep-1和Huh7细胞，发现细胞增殖活力降低、细胞周期进展阻滞、凋亡细胞增加、细胞迁移与侵袭能力下降。体内试验显示，干扰hsa_circ_0005785可抑制裸鼠HCC移植瘤的生长。hsa_circ_0005785主要定位细胞浆，经生信分析、RIP试验及荧光素酶试验证实，hsa_circ_0005785可直接绑定miR-578，且二者在HCC中表达呈负相关。经生信分析及荧光素酶试验证实，APRIL基因是miR-578靶基因，且二者在HCC中表达呈负相关。qPCR和Westernblot检测显示，hsa_circ_0005785可调控APRIL mRNA及蛋白的表达；同时，细胞功能试验显示hsa_circ_0005785可逆转miR-578对HCC生长与转移的抑制。提示hsa_circ_0005785/miR-578/APRIL信号轴在HCC的进展过程中起重要作用。通过本项目的研究，有望为 HCC 发病机制的探索提供新思路，为 HCC 的临床诊疗提供新靶标。