NQO1调控XIAP凋亡信号通路促进肝癌形成的作用机制研究

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death and the fifth-most-common cancer worldwide. Despite its high prevalence and inferior prognosis in general, our understanding of the pathogenesis of this cancer is very limited. Treatment of HCC is complicated by the fact that the disease is often diagnosed at an advanced stage when it is no longer amenable to curative surgery, and that current systemic chemotherapeutics are mostly inefficacious. A better understanding on the molecular basis of HCC is therefore urgently required to facilitate development of new therapeutic strategies. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a cytoplasmic 2-electron reductase implicated in the development of various cancer. Yet its role in carcinogenesis of HCC remains uncertain. We showed that NQO1 expression is upregulated in the majority of human HCC cell lines and tumor tissues. Functional studies revealed that kncokdown of NQO1 significantly suppressed proliferation and induced apoptosis in HCC cells that was associated with a remarkable downregulation of XIAP expression. Our preliminary data therefore revealed a novel role of NQO1 in the regulation of XIAP -mediated apoptosis, which plays a major role in HCC proliferation. The central theme of this proposal is thus to elucidate the molecular basis of NQO1 in regulating the XIAP-mediated pathway. We will also use animal model to evaluate if suppression of NQO1 will mitigate HCC growth. Therefore, our studies will provide new insights into the role of NQO1 in HCC tumorigenesis and may be helpful in developing NQO1 inhibitors as an effective treatment against HCC.

醌氧化还原酶1（NQO1）是一种依赖于还原型辅酶Ⅰ/Ⅱ的黄素蛋白酶，在多种肿瘤的发生发展中具有重要的作用，但在肝癌中的作用未明。我们的前期研究发现NQO1在肝癌细胞系和肝癌组织中表达明显增高，且NQO1敲减能抑制肝癌细胞增殖并显著诱导细胞凋亡，蛋白质质谱等分析发现NQO1在转录后水平调控凋亡抑制因子XIAP蛋白质的稳定性。据此，本项目提出假说：NQO1通过影响XIAP蛋白质的稳定性，调控下游凋亡信号分子活性，进而增强肝癌细胞抗凋亡能力，促进肝癌细胞恶性增殖。为此，本项目拟综合利用临床标本、细胞实验和动物实验，阐明NQO1在肝癌细胞恶性增殖中的作用；重点解析NQO1影响XIAP蛋白质的稳定性的分子机制；明确NQO1调控XIAP凋亡信号转导，促进肝癌形成的分子机理。本项目的实施，将有助于深入理解肝癌的发生机制，为寻找新的分子治疗靶点提供可靠的理论和实验依据。

肝细胞癌是全球死亡率排名第二的恶性肿瘤疾病，目前，肝细胞癌的致病机制不完全清楚，肝癌治疗的手段非常有限。NQO1是一种定位细胞质的氧化还原酶，在多种肿瘤组织中呈高表达。但NQO1在肝细胞癌发生发展中的作用机制尚不清楚，有待进一步研究。本研究通过分析公共数据库TCGA和GEO中多个数据集，发现肝癌癌组织中NQO1的mRNA水平明显高于癌旁组织，且NQO1的高表达与肝癌的肿瘤分级和预后生存率明显相关。从临床样本和肝癌细胞系中证实NQO1在癌组织和肝癌细胞系中的蛋白水平显著增高。通过构建NQO1敲减和过表达肝癌细胞系，研究证实NQO1基因敲减诱导肝癌细胞凋亡显著增加，抑制肝癌细胞增殖，而NQO1基因过表达则诱导肝癌细胞凋亡显著减少，显著增强肝癌细胞增殖。比较NQO1敲除的肝癌细胞和对照细胞中蛋白质的表达差异，发现凋亡抑制蛋白家族成员中XIAP蛋白质水平减少最为显著。在肝癌细胞中研究证实NQO1基因敲减对XIAP的mRNA水平无明显调节作用，但NQO1基因敲减可加速XIAP蛋白的降解，显著下调XIAP的蛋白质水平。在蛋白酶体受到抑制的NQO1基因敲减的肝癌细胞中XIAP蛋白水平又明显恢复。进一步研究证实NQO1基因低表达能显著抑制XIAP蛋白87位氨基酸位点的磷酸化。且通过检测肝癌组织样本证实NQO1蛋白水平与XIAP蛋白水平具有显著的正相关性。总的结果显示在肝癌细胞中NQO1高表达，可能通过增强XIAP蛋白磷酸化水平，上调XIAP蛋白水平的稳定性，抑制肝癌细胞的凋亡，促进肝癌的发生。本课题通过对NQO1在肝癌细胞中的功能鉴定和机制研究，阐明了NQO1在肝癌细胞中调控XIAP蛋白稳定性这一功能对维持肝癌细胞恶性增殖能力的分子基础。将有助于进一步理解肝癌的发生机制，同时将有利于发现新的肝癌诊断、治疗及预后相关因子，为开发NQO1抑制剂作为潜在的肝癌治疗手段提供有力的证据。