OGT介导的糖基化修饰对原发性肝癌免疫微环境中T细胞的调控作用与机制研究

Primary hepatocellular carcinoma (HCC) is a serious disease that presents significant harm to Chinese people. Although immune therapy has been applied to HCC, however, only part of the patient respond to it. The mechanism may relate to complicated immune microenvironment in HCC. More effective biomarkers for HCC immune therapy are needed. We found that OGT was upregulated in both HCC tumor tissue and serum. We determined that OGT induces endoplasmic reticulum (ER) stress through changing the lipid metabolism in HCC cells, thus activates downstream NF-κB pathway to promote HCC development. In addition, OGT participates in the activation of T cells under physiological condition. We also found that in HCC patients treated with interferon α(IFNα),OGT expression was downergulated compared with control group, and significantly upregulated in patients with HCC recurrence after IFNαtreatment. Nevertheless, the effect of OGT on T cells in HCC immune microenvironment under pathological condition still remains elusive. So we intended to analyze the O-GlcNAc modified proteins , genes and pathways regulated by OGT in T cells from HCC patients after IFNα or anti-PD-1 treatment, and verify them in in vitro and in vivo experiments. Our prospection is to illustrate the function and mechanism of OGT on T cells in HCC microenvironment, and to provide evidence for taking OGT as a biomarker for predicting effective immune therapy, prognosis and recurrence in HCC patients.

原发性肝癌是严重危害国人健康的重大疾病，免疫治疗虽已应用于肝癌患者，但仅部分患者有响应，其机理可能与肝癌复杂的免疫微环境有关，也亟需肝癌免疫治疗有效的生物标志物。申请人前期研究发现，OGT在肝癌组织和血清中升高，可通过改变肝癌细胞内脂质代谢，激活内质网应激及下游NF-κB通路，促进肝癌的发生发展。OGT参与生理状态下T细胞的活化，且在接受干扰素α治疗的肝癌病人血清中表达较对照组下降，而在治疗后复发病人血清中表达升高。但OGT对于病理状态下肝癌免疫微环境中T细胞功能的影响及机制尚未知。鉴于此，本项目拟分析接受干扰素及免疫治疗者肝癌组织中T细胞被O-GlcNAc修饰的蛋白及受OGT调控的基因及信号通路，并通过离体细胞和动物模型实验验证。本项目的实施预期将阐明OGT对肝癌免疫微环境中T细胞的作用及机制，初步将OGT作为免疫治疗有效或肝癌预后、复发风险的预测因子或生物标记物提供依据。

目的：我们对HCC肿瘤组织和癌旁组织进行了O-糖基化蛋白质组学测序，发现与癌旁组织相比，TRIM家族蛋白（tripartite motif-containing protein families）在HCC组织中受OGT的糖基化修饰。TRIM蛋白家族成员参与肝细胞癌(HCC)的发生发展。TRIM14对几种癌症有促进作用。本研究旨在探讨TRIM14在HCC中的作用及机制。.方法：通过检测TRIM14在肝癌组织及肝癌细胞株中的表达及在肝癌细胞系中建立TRIM14过表达或敲低模型，采用CCK8法、流式细胞术、Transwell法、RT-PCR、western blot、免疫荧光等方法验证了TRIM14对细胞增殖、化疗药物敏感性、凋亡、迁移、侵袭、自噬的影响。采用异种移植瘤模型验证TRIM14对肿瘤细胞生长的影响。.结果：肝癌患者肿瘤组织中TRIM14水平明显高于癌旁组织。TRIM14高表达患者的总生存率相对低于TRIM14低表达患者。TRIM14的上调增强了肝癌细胞的增殖、自噬、迁移、侵袭和化疗耐药性，减少了肝癌细胞的凋亡。TRIM14的敲除产生了相反的效果。在体内实验中，TRIM14上调促进了肿瘤生长。Western Blot结果显示，上调TRIM14可促进STAT3和HIF-1A的表达，下调TRIM14可抑制STAT3和HIF-1A的表达。此外，抑制STAT3和HIF-1A可减轻TRIM14对HCC细胞的促瘤作用。.结论:被修饰的TRIM14通过激活STAT3/HIF-1A轴促进肝癌恶性发展并诱导肝癌细胞的化疗耐药。此外，在临床患者中可将OGT作为α干扰素治疗 HCC 疗效的潜在生物标志物。