环状RNA circADAMTS13/miRNA/ADAMTS13信号轴在肝癌发生发展中的分子机制研究

ADAMTS13, a thrombospondin-1-type metalloprotease,could inhibit angiogenesis in hepatocellular carcinoma, but its up-regulation mechanism remains unclear. Our preliminary studies have identified specific circRNAs with potential roles in HCC development based on high-throughput sequencing technology. Expression spectrum and functional analysis revealed that one specific circRNA, defined as circADAMTS13, significantly lower expressed in HCC tissue and its overexpression in HCC cell lines could significantly inhibit cell proliferation and angiogenesis. Further studies indicated that circADAMTS13 could regulate the expression of its parental gene ADAMTS13, while the exact molecular mechanisms remained to be further explored. In this study, we will focus on circADAMTS13 / miRNA / ADAMTS13 signal axis, and further investigate the mechanism of circADAMTS13 inhibiting angiogenesis of HCC under inflammation induction. In addition, we will analyze the correlations between the expression of circADAMTS13 and ADAMTS13 in HCC tissues, and further explore their correlations with the clinical characteristics. This study will elucidate the molecular mechanisms of circADAMTS13 in hepatocarcinogenesis, and provide fundamental data and knowledges for applying the circADAMTS13 as a potential diagnostic and therapeutic target.

ADAMTS13是一种具有血小板反应蛋白1型基序的金属蛋白酶，可抑制肝癌的血管新生，但其上游调控机制目前仍不清楚。我们前期基于高通量测序技术筛选了肝癌发生发展相关的circRNAs并进行表达验证及功能研究，发现circADAMTS13在肝癌组织中低表达，且与肝癌细胞的增殖及血管生成相关。进一步分析发现其可调控其亲本基因ADAMTS13的表达，但作用机制仍有待探讨。本项目拟以circADAMTS13/miRNA/ADAMTS13信号轴为出发点，研究三者间的互作机制及在炎症诱导下circADAMTS13介导肝癌血管新生的分子机制；另外，本项目还将探讨circADAMTS13与ADAMTS13在肝癌组织中表达的相关性及其与肝癌临床特征的关系。本项目的研究将阐明circADAMTS13调控肝癌发生发展的分子机制，为其作为诊断和治疗的潜在靶标提供理论依据。

环状RNA（circular RNA，circRNA）作为一种广泛存在于真核生物转录组、具有组织细胞特异性的内源性非编码ncRNA逐渐被大家认识。circRNA可作为微小RNA(microRNA，miRNA)的海绵，抑制miRNA结合靶mRNA进而参与肿瘤发生发展。然而，肝癌进展过程中circRNA表达谱变化及其功能尚不清楚。本研究中我们使用二代高通量测序技术分析10例肝癌患者癌组织及其相应癌旁组织的circRNA表达谱变化差异。环状RNA 测序共鉴定到92204个环状RNA分子，其中大部分来源于外显子，长度大多在100-450个核苷酸左右，42个环状RNA在肝癌组织和癌旁组织中表达差异具有统计学意义，我们筛选出了差异表达最显著或者在测序过程中环化位点读数的数目最多的3种环状RNA（circADAMTS13、 circDPF3、circCASP8AP2），扩大26例样本进一步验证，circADAMTS13、circDPF3在肝癌组织中显著低表达与测序结果相符。其中肝癌组织中circADAMTS13 的低表达与较大的肿瘤直径和较晚的肿瘤BCLC分期相关。Kaplan-Meier生存分析表明低表达circADAMTS13 的患者具有更高的转移复发率，并且具有更差的总体生存率。稳定性实验证实circADAMTS13不受RNase R降解，比起其线性RNA更加稳定。在HepG2与PLC/PRF/5细胞中过表达circADAMTS13能够抑制肝癌细胞的增殖，并且促进肝癌细胞凋亡。荧光素酶报告系统结果显示circADAMTS13能与miR-484相结合，回复性实验进一步证实过表达miR-484可显著逆转circADAMTS13对肝癌细胞的抑制作用。总的来说，CircADAMTS13高表达与肝癌患者术后较好的预后相关，可作为预测肝癌预后的肿瘤标记物；CircADAMTS13可通过海绵作用结合促癌因子miR-484抑制肝癌细胞的增殖。此外，我们也发现另外一个环状RNA circEPB41L2在肝癌组织中显著也低表达，且与肝癌患者预后不良显著相关。其可以作为miR-590-5p的“海绵”进而调控肝癌细胞的增殖、侵袭和迁移。因此，CircADAMTS13和circEPB41L2有望成为新的HCC预后标志物和治疗靶点。