环状RNAcirc_103595/miR-185-3p/PLK1信号轴对肝癌不全消融后残余癌增殖侵袭的调控作用和机制研究

Radiofrequency ablation (RFA) is one of the most essential treating strategies for liver cancer. The rapid progression of residual tumor after insufficient RFA seriously affects the prognosis of patients, and the exact mechanism is still lack of elucidation. The regulatory mechanism of circular RNAs in residual tumor has not been reported. Previously, we have established the expression profiles of noncoding RNA in residual tumor after insufficient RFA by using RNA-sequencing. Differentially expressed circular RNA circ_103595 was validated by clinical samples. We also found that circ_103595 might influence the expression of PLK1 by regulating miR-185-3p. Furthermore, close targeting correlations existed among circ_103595, miR-185-3p and PLK1, which could play an essential role in the proliferation of residual tumor. Further pathway analysis revealed that cell cycle pathway was the most significantly enriched pathway affected by circ_103595/miR-185-3p/PLK1 axis, which was essential for the rapid proliferation of residual tumor. On this basis, this project proposes to evaluate the prognostic value of circ_103595/miR-185-3p/PLK1 axis for patients with liver cancer based on a large clinical sample size. Meanwhile, based on the cell and animal models, we aim to further explore the role of circ_103595 in the proliferation and invasion of residual tumor, unraveling the effective pathways and targets in the downstream regulations.

射频消融作为肝癌主要治疗手段之一，不全消融导致残余癌的快速进展严重影响患者预后，而对其机制一直未能清楚阐明。环状RNA在不全消融残余癌的机制研究未见报道。申请者在前期工作中通过高通量测序建立了肝癌射频消融残余癌的特征非编码RNA表达谱，在差异表达的环状RNA中，circ_103595得到了临床样本的验证。并根据表达情况及碱基互补配对序列发现circ_103595可通过调控miR-185-3p来影响其靶基因PLK1的表达，从而介导肝癌的增殖。进一步分析表明细胞周期是该信号轴影响最显著的通路，故假设circ_103595/miR-185-3p/PLK1信号轴对残余癌快速增殖至关重要。本项目拟在此基础上，从临床大样本中分析总结circ_103595/miR-185-3p/PLK1信号轴与肝癌临床相关性以及对射频消融术预后判断价值。同时，在细胞和动物模型中进一步深入探讨其在残余癌中的调控作用。

射频消融（RFA）是目前最常见的热消融治疗技术之一，是肝癌的微创治疗方式。既往一项临床研究表明，RFA是一种安全技术，可作为早期肝癌的一线治疗，接受RFA的患者总5年生存率为67.9%。由于RFA是一种热消融治疗，肿瘤区域内或周围血管中的血流会带走热量，引起散热器效应，限制凝血坏死的程度，从而导致消融不全和局部肿瘤复发。这是影响患者临床结局的重要因素。更重要的是，先前的研究发现，不完全的RFA会导致侵袭性肿瘤复发，导致预后较差。因此，鉴定不完全RFA的生物标志物对于肝癌的早期确定和治疗至关重要。环状RNA在不全消融残余癌的机制研究未见报道。RFA处理肝癌移植瘤后，经全基因组测序检测到76个差异性circRNA，其中21个上调，55个下调。计算生物学分析表明，T细胞受体信号通路是与改变的circRNA相关基因中最显着的富集通路，包括LCK，AKT3和DLG1的富集。在差异表达的circRNA中，我们对circ_103595临床样本以及后续的功能验证。实时定量PCR表明hsa_circRNA_103595在不全消融后的肿瘤组织中显著上调，而功能实验的结果的也表明hsa_circRNA_103595的敲低会显著抑制肝癌细胞的增殖、迁移与侵袭。蛋白免疫印迹实验同时提示，hsa_circRNA_103595的水平会促进上皮细胞-间充质转化的进展，从而影响细胞的迁移与侵袭能力。双荧光素酶实验证明，hsa-miR-185-3p是hsa_circRNA_103595的靶miRNA，hsa-miR-185-3p同时也调控着PLK1分子的表达。因此，hsa_circRNA_103595通过吸附hsa-miR-185-3p从而调控PLK1的竞争内源性RNA机制或许是消融后残余癌快速进展的关键因素之一。