影像引导热消融对肝癌ZNF545表观遗传学改变的调控作用

Tumor recurrence and metastasis is the bottleneck of restricting the effect of image-guided thermal ablation of hepatocellular carcinoma(HCC). Reducing the intra-hepatic and extra-hepatic metastasis at the same time of improving the ability of in situ inactivation is one of the key problems that need to be resolved for HCC ablation. Epigenetics change has been definited as the main way of HCC oncogene activation and tumor suppressor gene inactivation. Thermal field of ablation can promote the body's immune function during eliminating tumor load. Based on this internal environment change, it is worthy to explicit that if the methylated tumor suppressor gene before ablation is associated with HCC recurrence and if its methylation after ablation restores its anticancer activity. Our preliminary study found transcriptional regulation gene ZNF545 occurs methylation frequently in liver cancer cell lines and tissues, therefore it is expected to become sensitive marker for detecting HCC epigenetic changes. This project intends to analyze the expression and DNA methylation of ZNF545 before and after thermal ablation in liver cancer cell lines and HCC tissue and peripheral blood by methylation specific PCR technology, to clear the regulation effect of thermal ablation on ZNF545 epigenetics change and the mechanism of ZNF545 methylation on HCC recurrence after ablation. The study will lay the foundation for the platform of prospective, predictable individual ablation and will provide the biological evidence for the combination of ablation with epigenetics therapy to improve the HCC treatment effect.

肿瘤复发转移是制约影像引导热消融治疗肝癌疗效的瓶颈，提高原位灭活能力的同时降低肝内外转移是肝癌消融需要解决的关键问题之一。表观遗传学改变已被明确是肝癌癌基因激活和抑癌基因失活的主要途径。消融热场在消灭肿瘤负荷的同时可以提升机体免疫功能，在此内环境改变基础上，热消融前甲基化的抑癌基因是否与肝癌复发相关且消融后是否发生去甲基化而恢复其抑癌活性值得明确。本课题组前期研究发现转录调控基因ZNF545在肝癌细胞系和组织中频繁甲基化，有望成为检测肝癌表观遗传改变的敏感指标。本项目拟采用甲基化特异性PCR技术分析肝癌细胞系及HCC热消融前后组织和外周血中ZNF545基因的表达和DNA甲基化改变，从而揭示消融热场对ZNF545表观遗传学改变的调控规律，并阐述ZNF545甲基化对肝癌消融后复发转移的作用机制，为建立前瞻性、可预测的个体化消融平台奠定基础，并为提高肝癌消融疗效及联合表观遗传治疗提供生物学依据。

肿瘤复发转移是制约影像引导热消融治疗肝癌疗效的瓶颈，提高原位灭活能力的同时降低肝内外转移是肝癌消融需要解决的关键问题之一。表观遗传学改变已被明确是肝癌癌基因激活和抑癌基因失活的主要途径。热消融前甲基化的抑癌基因是否与肝癌复发相关且消融后是否发生去甲基化而恢复其抑癌活性值得明确。本课题对入组HCC患者进行超声引导下微波或射频消融治疗，留取治疗前患者外周血、瘤周肝组织和HCC组织、治疗后不同时期外周血及对照正常肝标本，并培养7株肝癌细胞系，采用反转录PCR(RT-PCR)、甲基化特异性PCR (MSP)、Sequenom甲基化检测、免疫组化、染色体免疫沉淀等技术分析肝癌细胞系，组织及外周血标本中ZNF545基因的表达和DNA甲基化改变，结合患者消融后复发转移生存信息，开展以下研究：(1)分析ZNF545在肝癌中表观遗传学改变的诊断价值及在肝癌发生发展进程中的作用；(2)分析消融热场在消灭HCC肿瘤负荷和提高机体免疫力后对ZNF545甲基化和表达调控的影响；(3)分析ZNF545甲基化与HCC超声引导热消融治疗后转移复发的相关性及其对HCC生存预后的影响。通过细胞增殖实验、克隆形成实验、细胞凋亡实验证明ZNF545显著抑制肝癌细胞增殖和克隆形成能力，促进肝癌细胞凋亡，是潜在的肝癌抑癌基因。进一步将基础研究结果应用于临床，通过微波消融101例肝癌患者并对患者ZNF545甲基化检测，平均随访观察28.7个月，发现ZNF545高甲基化组具有更差的生存预后(p= 0.041)，说明该基因是预测肝癌消融预后的有价值基因标志。但对20例患者不同疾病时期的血液样本甲基化检测尚未得出阳性结果。本项目阐述了ZNF545甲基化对肝癌消融后复发转移的作用机制，为建立前瞻性、可预测的个体化消融平台奠定基础，并为提高肝癌消融疗效及联合表观遗传治疗提供了生物学依据。