乙酰化修饰介导抑癌基因ZHX2胞核定位异常在肝细胞肝癌中的作用及分子机制研究

Aberrant expression and regulation of tumor suppressors are the key factors of tumorigenesis, therefore, it’s important to investigate the mechanisms about abnormal regulation of tumor suppressors. ZHX2, as a transcriptional factor, modulates abundant genes, involving in development of various diseases and attracting extensive attention. The previous study from applicant’s lab demonstrated ZHX2 as an indispensable tumor suppressor in hepatocellular carcinoma (HCC). The aberrant subcellular location of ZHX2 is a vital reason for liver carcinogenesis, however, the mechanism still remains elusive. Our preliminary study identified functional sequence of ZHX2 nuclear location signal (NLS) and illustrated that mutation of genes in NLS was not the main reason of abnormal location of ZHX2 in liver cancer cells. The mass spectrometry analysis combined with immunoprecipitation showed that ZHX2 in liver cancer cells contained several acetylated amino sites and pan-acetylation was significantly increased. Therefore, it’s hypothesized that acetylation of ZHX2 led to disordered cytoplasm-nuclear location and deprivation of tumor suppressor-function. The project here is designed to define that acetylation is the key factor to disturb entry of ZHX2 into nuclear and function in HCC, and explain the involving molecular mechanisms, using multiple in vivo and in vitro assays combined with clinical studies. This program will provide new direction for elucidating mechanisms about malfunction of tumor suppressor in advancement of HCC, and novel targets for anti-HCC therapy as well, which is of great significance.

抑癌基因的异常表达和调控是肿瘤发生发展的关键因素，研究抑癌基因异常调控机制具有重要意义。ZHX2作为转录因子调控多种关键基因，参与不同疾病进展，受到越来越多的关注。实验室前期研究揭示，ZHX2是肝细胞肝癌中重要的抑癌基因，其胞核定位异常是肝癌发生发展的关键因素，但其调控机制尚不明确。申请人近期鉴定了ZHX2核定位信号关键序列，测序结果提示，核定位信号基因突变并非ZHX2核浆定位异常的主要因素；进一步质谱及免疫沉淀实验显示，ZHX2蛋白含多处乙酰化修饰位点，且泛乙酰化水平明显升高。据此提出假说：肝癌细胞内ZHX2乙酰化修饰导致其核浆定位紊乱，抑癌基因功能丧失。本项目拟利用体内外实验和临床研究，综合应用多种技术手段，明确肝癌细胞内乙酰化修饰是影响ZHX2入核发挥生物学功能的主要因素，并阐明分子机制。本研究将为揭示肝癌进展中抑癌基因功能异常的机制提供新角度，为抗肝癌治疗提供新靶点，具有重要意义。

在我国，肝癌是发病人数排名第四，死亡人数位居第二的肿瘤疾病，肝细胞肝癌约占其中80%。乙肝病毒慢性感染是肝细胞肝癌的主要诱发因素，非酒精性脂肪肝炎正逐渐成为另一重要危险因素。在肝细胞肝癌的发生及进展过程中，伴随着大量促癌基因或抑癌基因表达水平及功能的变化。ZHX2是广谱表达于多种组织细胞的转录因子，在肝癌中发挥抑癌基因的功能。本课题组前期研究揭示，乙肝病毒感染或高脂因素均可引起肝癌细胞中ZHX2表达水平明显降低。值得注意的是，在临床标本中，与癌旁组织相比，肝癌细胞核内ZHX2的表达水平显著下降，而细胞内总表达量无明显差异。这提示，ZHX2在肿瘤细胞内可能核浆定位异常并导致其无法发挥生物学功能，另外，其中机制尚不清楚。为明确上述问题，本项目首先在前期研究中确定了介导ZHX2进入细胞核的核定位信号的关键序列及位点，并验证了核定位信号异常可影响ZHX2的抑癌功能及抑制HBV复制功能。同时，通过临床肿瘤标本排除了核定位信号区域DNA突变作为ZHX2异常核浆定位主要原因的可能性。在上述基础上，研究利用系列体外实验发现，核定位信号419位点赖氨酸乙酰化修饰是引起ZHX2胞浆滞留的主要原因。通过免疫共沉淀实验和生物素标记手段确定了，ZHX2主要通过与输入蛋白KPNA2结合，并在其介导下进入细胞核，而核定位信号419位点乙酰化修饰则影响了ZHX2与KPNA2的结合，导致其胞浆滞留。进一步通过筛选多个乙酰基转移酶，明确了核定位信号419位点的乙酰化修饰主要由GCN5催化，敲低GCN5表达或加入特异性抑制剂能够促使ZHX2进入细胞核。综上所述，本研究首次阐明了，肝癌细胞内GCN5介导ZHX2核定位信号419位点发生乙酰化修饰，进而影响ZHX2与输入蛋白KPNA2的结合，引起核浆定位异常，导致其无法在核内发挥生物学功能。这为探索新型抗肝癌药物靶点提供了理论依据。在计划任务之外，本项目还参与探究了，ZHX2通过调控脂质代谢抑制肝癌细胞生长，以及CD169阳性巨噬细胞协同增强射频消融治疗肝癌效果的研究。