超级增强子调控GATA4对肝癌循环肿瘤细胞恶性生物学行为的作用机制研究

Hepatocellular carcinoma tends to disseminate circulating tumor cells (CTCs) into the blood, which is the key of metastasis. However, the mechanism by which CTCs survive in circulation and implant metastasis is still underdetermined. Using a novel CTCs-3D culture system, we previously found CTCs from HCC patients presented two types with widely different ability of proliferation and invasion, which we named as multi-cellar clone (MC) and oligo-cellar clone (OC). Single-cell sequencing analysis revealed GATA4 was overexpressed specifically in MC. We used H3K27ac ChIP-Seq data to identify extensive acetylation of histone H3 lysine 27 (H3K27ac) on the upstream of GATA4, which defined the active super-enhancers (SEs). Therefore, we proposed the hypothesis that the SEs induced the overexpression of GATA4 in MC, which in turn affected the phenotype and metastasis of HCC-CTC. In this study, we will find how the specific SEs regulate GATA4 and affect the biological malignant behaviors of CTCs. We will use CRISPRi/a techniques to disturb the SEs, and determine the effects of expression of GATA4 on phenotypes, anti-anoikis and clone formation of CTCs, as well as the effects of GATA4 cis element and downstream genes on secondary tumorigenicity of CTC. Based on HCC-CTC 3D culture and in vivo tumorigenicity experiments, this study will explore the effects of SEs-regulated GATA4 on HCC invasiveness, illuminate the molecular mechanism of HCC-CTC heterogeneity from a new perspective, and find new targets for treatment strategies on HCC metastasis.

肝细胞癌易形成循环肿瘤细胞（CTC），是导致继发转移的关键。但CTC失巢进入循环存活并种植形成转移灶的机制尚不明了。前期我们通过肝癌CTC-3D培养发现，CTC存在增殖和侵袭能力迥异的两种类型即多细胞（MC)和寡细胞型（OC)；单细胞测序发现GATA4分子在MC中特异性高表达。通过ChIP-Seq发现GATA4上游存在活跃的超级增强子（SEs），可能与GATA4异常高表达及MC的侵袭性密切相关。基于此，本课题拟研究SEs调控GATA4及其与肿瘤恶性生物学特性形成的分子机制；通过CRISPRi/a技术干预SEs活性，研究GATA4表达与CTC表型、抗失巢凋亡和克隆形成等功能的关系，分析GATA4顺式元件和下游基因对CTC继发成瘤的影响。采用3D培养体系及体内成瘤实验研究SEs调控GATA4对肿瘤侵袭性的影响，从新的角度阐明CTC异质性的分子机理，以期发现针对HCC转移治疗的新靶点和方法。

循环肿瘤细胞（CTC）是肝细胞癌（HCC）根治术后发生肿瘤复发和转移的关键，我们通过前期HCC-CTC 3D培养发现，多细胞型克隆（MC）CTC具有极强的侵袭性和成瘤能力，单细胞测序显示GATA4分子在MC中特异性高表达，而ChIP-Seq发现GATA4上游存在活跃的超级增强子（SEs）。通过本研究进一步阐明了CTC在HCC复发和转移中发挥“种子”作用的分子机理，高表达GATA4的肝癌细胞系中lncRNA SNHG8显著高表达, 而SNHG8 通过对miR-149的吸附作用，对抗miR-149的肿瘤抑制作用，使HCC细胞活性和侵袭能力显著增强。研究过程中进一步探索了合适的“土壤”即肿瘤微环境在“种子”定植中的关键作用，肝癌细胞、炎性细胞以及抑制性细胞因子共同构成了肿瘤微环境中复杂的免疫抑制网络，HCC肝移植术后高复发风险的患者术前即处于一定程度的免疫抑制状态，表现为Treg和Th2比例的增加和IL-10水平的升高，以及IFN-γ和IL-2水平的降低。此外，我们发现GPC3分子在MC型克隆中显著高表达，成功构建并优化了靶向于GPC3的CAR-NK，为肝癌根治术后低肿瘤负荷患者针对CTC的免疫治疗提供了新的思路，有待于进一步研究。