长链非编码RNA HOTTIP调控肝癌复发转移的作用及表观分子机制

The long-term survival of patients with hepatocellular carcinoma (HCC) following surgery remains unsatisfactory due to the high frequency of recurrence. Therefore, a better understanding of the molecular mechanisms underlying HCC recurrence becomes the key issue in developing effective therapeutic strategies. We aim to clarify the role and underlying epigenetic mechanisms of the novel long non-coding RNA HOTTIP in the regulation of HCC recurrence and metastasis. We examine the expression patterns of HOTTIP and analyze the correlation of the abnormal expressions of HOTTIP with clinico-pathological characteristics and recurrence of HCC, as well as the prognosis of the patients. Liver cancer cells stably expressing or interfering HOTTIP expression are eatablished. The effects of HOTTIP overexpression or depletion on HCC cells are determined by viability, apoptosis, migration and invasion assays and observed its tumorigenesis and metastasis in nude mice. Changes in gene expression and signaling pathways induced by HOTTIP knockdown are identified by microarray and immunoblot analyses. The epigenetic mechanisms of HOTTIP are explored by RNA immunoprecipitate, RNA pulldown and chromatin immunoprecipitation analyses. This project will explore the role of HOTTIP in the development and progression of HCC, and reveal its cellular and molecular mechanism underlying, thus contributing to provide a new target for the treatment of HCC and an important biomarker for the evaluation of prognosis of HCC patients following surgery.

肝癌术后肿瘤复发是制约患者长期生存的关键，揭示肝癌复发转移的分子新机制，建立合理有效的干预策略具有重要意义。本课题将系统研究新型长链非编码RNA HOTTIP在调控肝癌复发转移中的作用及表观分子机制。分析肝癌组织中HOTTIP基因的表达水平与肿瘤临床病理特征和预后之间的关系；采用分子克隆技术建立稳定高/低表达HOTTIP的人肝癌细胞株，应用肝癌细胞体外模型和裸鼠肝癌复发转移模型，明确HOTTIP的表达变化对肝癌细胞增殖、凋亡、迁移及侵袭能力、成瘤性及肿瘤转移等恶性生物学行为的影响；采用全基因组表达谱芯片，深入研究HOTTIP的表达变化对Wnt5a通路等下游分子表达和功能的影响，同时应用RNA免疫共沉淀、RNA pulldown及CHIP等技术阐明其调控Wnt5a的表观分子机制。本项目对深入阐明HOTTIP在肝癌复发转移中的功能及机制，寻求肝癌复发新型分子标志物及潜在治疗新靶点具有重要意义。

肝癌术后肿瘤复发是制约患者长期生存的关键，揭示肝癌复发转移的分子新机制，建立合理有效的干预策略具有重要意义。本课题前期通过10对肝癌与癌旁组织二代测序分析，筛选并确定新型长链非编码RNA HOTTIP为潜在的促癌基因。本研究通过系统研究新型长链非编码RNA HOTTIP在调控肝癌复发转移中的作用及表观分子机制，为肝癌复发新型分子标志物及潜在治疗新靶点提高新思路。.在本研究中，我们评估了HOTTIP的表达模式以及其表达量在预测肝移植术后肝癌患者肿瘤复发率的临床意义。对200例肝癌标本中HOTTIP表达量进行检测，并进行其与病人预后、临床指标的相关性分析。利用siRNA敲低HOTTIP表达来探讨其在肿瘤进展中起到的作用。结果提示HOTTIP在癌组织中的表达水平明显高于癌旁组织中的表达。多因素分析显示，HOTTIP高表达是肝癌患者肿瘤复发和较低总生存时间的独立危险因素。即时是不符合米兰标准的低HOTTIP表达量肝癌患者在肝移植后依旧呈现较对较低的无复发生存率和总生存率。此外，在肝癌细胞系中敲低HOTTIP降低了肝癌细胞的侵袭率以及增加了化疗敏感性。再者，HOTTIP SNP-rs2071265与HCC患者的早期复发相关，影响HOTTIP表达。总之，在HCC的HOTTIP表达水平可以成为一个可预测肝癌患者肝移植术后预后的候选生物标志物。此外，HOTTIP可能是一个潜在的治疗靶点。.