COMMD7与NF-κB之间的正反馈互调控：COMMD7基因在肝癌细胞中异常过表达的分子机制研究

Our previous studies over the past few years have firmly established COMMD7, a newly identified gene in liver cells by our research group in 2005, as an oncogene which when overexpressed promotes hepatocellular carcinoma (HCC) by favoring proliferation, metastasis and invasion, reducing apoptosis, and developing multi-drug resistance in hepatoma cells. In the contrary, silencing COMMD7 yields a strong anti-HCC efficacy both in vitro and in vivo. Clearly, interrogating the molecular mechanisms underlying the overexpression of COMMD7 is a necessary step towards complete understanding of the oncogenic effect of this gene, and a highly desirable task for developing a new anti-HCC strategy using COMMD7 as a therapeutic target as well. Our further research has also revealed that COMMD7 acts through stimulating NF-κB activities. Most recently, our pilot experiments have provided us the data strongly suggesting the potential of NF-κB to transcriptionally activate COMMD7 expression. These previous studies and preliminary results prompted us to hypothesize that between COMMD7 and NF-κB there is a positive feedback circuitry mechanism: during cancerigenesis, enhanced NF-κB activation stimulates COMMD7 transcription that in turn further augments NF-κB activities to promote COMMD7 transcription, eventually leading to COMMD7 overexpression and NF-κB hyper-activation. To test this hypothesis, we plan to first conduct bioinformatics analysis to computationally predict the promoter region of the COMMD7 gene and the cis-acting elements within the region, particularly those for NF-κB binding. Then functional genomics technologies will be employed to experimentally identify the transcription start site, to characterize the core promoter region, and to verify the ability of NF-κB to interact with its binding sites. Next, we will manage to normalize the expression or correct the overexpression of COMMD7 via specifically interfering the binding of NF-κB to its cis-acting element in the COMMD7 promoter by means of triplex-forming oligodeoxynucleotides (TFO) techniques. And finally, TFO will be examined for its potential to minimize the gain of the positive feedback loop so as to produce anti-HCC efficacy against cell proliferation and multi-drug resistance in HepG2 cells and xenograft tumor growth in nude mice.

肝癌是我国高发生率和高死亡率的癌症之一。我们前期研究揭示新基因COMMD7的异常过表达对肝癌增殖、侵袭、转移和发展多药耐药性均起着促进作用，沉默此基因则产生抗肝癌效应，但其过表达的分子机制尚不明确。我们发现COMMD7的促肝癌作用是通过激活NF-κB来实现的；而NF-κB作为转录因子又可能反馈激活COMMD7的表达。我们由此提出一个假说：在癌变时NF-κB转录上调COMMD7，后者又反馈激活NF-κB，活化的NF-κB进一步上调COMMD7使之过表达。在NF-κB与COMMD7之间存在着一个正反馈调控环路，维持COMMD7的异常过表达和NF-κB的"功能亢进"。本研究拟验证此假说：分析COMMD7功能基因组学特性，阐明NF-κB转录调控COMMD7的分子机制和两者之间的正反馈互动关系,并探讨通过应用三链形成寡核苷酸技术靶向干扰此正反馈环路来逆转COMMD7过表达从而治疗肝癌的可能性。

肝癌是发病率和死亡率较高的恶性肿瘤，前期研究发现COMMD7在肝癌组织/细胞中高表达，具有促进肝癌细胞增殖、侵袭转移以及多药耐药等功能，但具体调控机制尚不明确。通过三年的研究，本项目完成了以下研究目标。1.揭示了肝癌中COMMD7与NF-κB正反馈调节关系及其促癌机制：研究发现肝癌中COMMD7的表达水平与NF-κB的表达呈正相关，二者之间存在正反馈调节，另外研究还发现COMMD7可调控CXCL10/ CXCR3的表达从而促进肝癌发生；2. 解析了肝癌细胞中NF-κB转录调控COMMD7基因异常过表达的机制：生物信息学方法预测并通过ChIP和EMSA实验验证了NF-κB与COMMD7基因启动子上4个核心位点的结合；3. 探索了COMMD7在肝癌干细胞中的表达情况及其生物学功能：研究发现COMMD7在肝癌干细胞中高表达，发挥促进肝癌干细胞增殖，抑制分化的功能；4. 确定了COMMD7在胰腺导管腺癌中的表达模式、临床意义和生物学功能，并初步阐述了调控机制：研究发现COMMD7在胰腺导管腺癌组织/细胞中高表达，其表达水平与肿瘤分化程度及肿瘤分期相关，是胰腺导管腺癌患者预后的独立危险因素，通过调节ERK1/2的表达调控胰腺导管腺癌细胞增殖和侵袭转移。研究结果表明，COMMD7在维持肝癌和胰腺导管腺癌恶性生物学行为中发挥重要作用，可能是治疗肝癌和胰腺导管腺癌的潜在分子靶点。.本项目目前已取得的成果有：1. 以第一作者或通讯作者在国外SCI收录期刊发表论著6篇，最高IF: 5.165；2. 作为导师完整培养研究生2名（其中1名为课程班学员），协助科室培养硕士研究生2名，博士研究生1名；3. 结合前期研究成果，申报并获得重庆市科技进步二等奖1项。