癌旁肝细胞表达/分泌IL-2调节炎症免疫微环境抑制肝癌转移复发的分子机制

Our previous studies confirmed that IL-2 in peritumoral microenvironment played an important role in inhibiting metastasic recurrence of hepatitis B-related hepatocellular carcinoma. The patients with high IL-2 level in peritumoral tissues have good prognosis usually. Moreover it was initially proved that the IL-2 was mainly from peritumoral hepatocytes, and correlated with hepatitis B virus inflammation. In this subject, we will use in vitro cell culture, clinical liver tissue samples and molecular biology and immunological technology: (1) analyzing IL-2 expression by hepatocytes in protein and transcription levels and intracellular location in ultra structure levels; (2) investigating initiate link and downstream signal pathways and key molecules involved in expression/secretion procedure of IL-2 in hepatocytes; (3) studying molecular mechanism for hepatocytes regulating target cells (TAM, Th1/Th2, CD4/CD8 and CTL/Treg) in peritumoral microenvironment via IL-2 and then inhibiting invasion and metastasis of tumor. Resolution of these problems will allow us to recognize hepatocytes' biological function in the invasion and metastasis of tumor from new perspective. And this will contribute to development of new immune regulation strategy preventing metastatic recurrence of liver cancer. There have been no domestic and foreign reports focused on hepatocytes' role in metastasis and recurrence of tumor so far.

我们前期研究证实癌旁肝组织中的IL-2在抑制乙肝相关性肝癌的转移复发中发挥重要作用, IL-2高表达的患者预后通常好，并初步证实其主要来源于肝细胞,与乙肝病毒（HBV）慢性感染有关。本课题将应用HBV刺激和分子免疫学等技术在体外培养细胞系和临床样本中:(1)蛋白和转录水平分析肝细胞受HBV影响表达IL-2,并细胞内超微结构定位；(2)研究肝细胞表达/分泌IL-2的启动和下游信号通路及调控分子，明确调节肝细胞表达IL-2高低的限速环节或关键分子；(3)分析肝细胞通过IL-2对免疫微环境中与转移复发密切相关的TAM细胞、Th1/Th2、CD4/CD8和CTL/Treg平衡的调节作用。这些问题的解决将使我们从新视角认识癌周肝细胞在肿瘤侵袭转移中的生物学作用，有助于发掘防治肝癌转移复发的新型免疫微环境调变策略，目前针对肝细胞在抗转移复发中的作用国内外均未见起步性研究报道。

癌旁肝组织中的IL-2在抑制乙肝相关性肝癌的转移复发中发挥重要作用。本课题证实了癌周组织的肝细胞表达IL-2与乙型肝炎病毒（HBV）的慢性感染有密切相关性。并进一步发现HBV病毒的x抗原（HBx）以及微环境的较低氧化压力能诱导正常肝细胞在体外表达IL-2，并且这一过程由MAP3K7/NF-kB通路介导，IL-2主要表达于肝细胞胞浆。肝细胞表达IL-2促进癌周CTL/Treg比例增高，使免疫反应向Th1型发展，有利于肝癌侵袭转移的抑制。这些结果提示慢性炎症及氧化压力微环境对肝癌转移复发的重要性，在此基础上我们进一步发现了与IL-2表达相关的一种巯基氧化酶(QSOX1)，在体外细胞实验和体内裸鼠模型实验中发现并证实了QSOX1促进肝癌细胞凋亡和抑制肝癌细胞侵袭转移的生物学功能。大样本免疫组化分析证实癌及癌旁组织QSOX1是肝癌患者术后生存以及复发的独立预后因素。并进一步发现QSOX1抑制肝癌细胞中Integrinβ1/FAK 和 EGFR/Raf/ERK 通路。质谱分析确定血清中QSOX1主要是核心岩藻糖基化蛋白，之后在肝癌患者血清样本中证实血清核心岩藻糖基化的QSOX1是潜在的预测肝癌患者术后转移复发的分子标志物。最后我们通过基因定点突变技术在体外细胞实验和体内裸鼠模型实验中发现QSOX1行使抑制肝癌细胞侵袭转移的功能依赖于130氨基酸位点的核心岩藻糖基化。本课题研究结果提示有必要进一步探索调节和控制肝癌微环境的氧化/抗氧化平衡来寻找干预治疗肝癌转移复发的方法；开发外源局部给予QSOX1可以作为干预治疗肝癌转移复发的潜在方法。