早发肝癌高频整合位点HBx-SINE的垂直传递及其促进肝癌发生的分子机制

Compared with late-onset hepatocellular carcinoma（HCC）, Early-onset HCC (< 40 years old) is more malignant, more insidious and more difficult to detect. Limited by clinical samples and research methods, there are few studies focused on early-onset HCC. Our pilot study identified a high frequency HBV integration site, HBx-SINE， in early-onset HCC by using virus capture sequencing methods. We further detected HBx-SINE in normal tissues adjacent to tumor and peripheral blood mononuclear cells (PMBC), suggests the integration site may be not a somatic integration, but a germline integration. Based on these observations, we speculate that after HBV infection germ cells, HBV integrated in these cells, consequently, the HBV integration sites passed to the offspring embryo cells, so that the child was born carrying a "first hit", thus with a earlier onset HCC. The project will further Confirmed the hypothesis by: expanding clinical samples to validate HBx-SINE detection earlier-onset HCC, use of cell and animal experiments to illustrate HBx-SINE up-regulating MYC and PVT1 expression and in turn, promote the development of early-onset HCC. Finally, we use of abandoned germ cell or embryo to confirm HBV vertical transmission characteristics of HBV integration, thus finally clarify vertical transmission of HBV integration and the role and mechanisms of HBV integration in the early-onset HCC. Our work will provide new target for early-onset HCC detection and treatment. We also provide a new theoretical basis for HBV integration in early-onset HCC.

早发肝癌（发病〈40岁）恶性程度更高、起病更隐匿、临床更难发现。囿于样本和技术手段限制, 目前针对早发肝癌研究较少。我们前期利用病毒捕获测序发现，8q24MYC/PVT1增强子区存在早发肝癌高频整合位点HBx-SINE，整合后上调MYC/PVT1表达。且HBx-SINE在癌旁和外周血单个核细胞（PMBC）中均能检出，提示其可能为生殖系整合。由此推测：HBV感染生殖细胞后发生整合，整合片段如HBx-SINE通过生殖细胞传递给子代，使子代出生即携带“第一次打击”，因而发病年龄更早。本项目将进一步扩大临床样本验证HBx-SINE在早发肝癌中的作用；利用细胞和动物实验阐明HBx-SINE通过激活增强子上调MYC、PVT1、进而促进肝癌早发的分子机制；利用FISH等获得HBV整合子代传递的直接证据，最终阐明HBV整合通过垂直传递促进肝癌早发的作用机制，为早发肝癌诊断与个体化治疗提供分子靶点和理论基础

本项目深入探讨了HBx-SINE通过结合PARP1而增强c-Myc表达的机制，验证了我们标书中的科学假设：HBx-SINE整合位点可能通过HBV的增强子/HBx启动区募集转录因子，进而激活c-Myc，上调c-Myc的表达。同时，我们对于HBx-SINE激活c-Myc的上调表达的机制进行了深入研究，肿瘤组织中，人染色体HBV高频整合8q24区整合序列HBx-SINE通过结合PARP1增强c-Myc启动子在HBV-HCC细胞中的转录调控活性而导致肿瘤组织中c-Myc高表达促进HBV-HCC早期发生。进一步对6对早发性HBV-HCC标本进行三代测序，发现在早发性HBV-HCC患者肿瘤组织中HBV整合呈克隆性，提示HBV整合优势克隆扩增为HBV-HCC的早期驱动事件。HBV-HCC 患者肿瘤组织中HBV促进了基因组的不稳定，导致不同染色体重组率增加，可能促进HBV-HCC 的早期发生。HBV整合与基因组拷贝数变异存在关联。这些研究为HBV-HCC的诊断和治疗提供新的分子靶点和思路。