Liu Xing, Cui Shu-Jian, Zhu Shi-Jun, Geng De-Chun, Yu Long
State Key Laboratory of Genetic Engineering, Fudan University Shanghai, China ; National Engineering Center for Biochip at Shanghai Shanghai, China.
College of Bioscience and Biotechnology, Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Yangzhou University Yangzhou, China.
Int J Clin Exp Pathol. 2014 Apr 15;7(5):2044-55. eCollection 2014.
Neurite outgrowth inhibitor proteins (Nogos) comprise a family of three major members and are characterized by a conserved RHD domain. Among all the members, Nogo-B was identified to be significantly elevated and to play an important role in liver cirrhosis while Nogo-C was the shortest one and received little attention. The aim of this study is to investigate the relevance and mechanism of Nogo-C involved in Hepatocellular carcinoma (HCC).
The expression of Nogo-C in paired HCC specimens was measured with quantitative RT-PCR. The function of Nogo-C over expressing in SMMC-7721 and WRL-68 HCC cell lines were estimated through cell proliferation assay and colony formation assay. A proteome-wide identification of Nogo-C-binding proteins was performed using affinity purification combined with a highly sensitive mass spectrometric technique. The protein interactions were confirmed using co-IP and immunofluorescence confocal assays.
Compared with the neighboring pathologically normal tissues, the expression of Nogo-C mRNA was extremely down-regulated in HCC specimens and was significantly related to greater tumor size and worse prognosis. Overexpression of Nogo-C in HCC cell lines resulted in an inhibition of cell growth. A total of 73 proteins were detected and considered in association with Nogo-C, among which B-raf and Nogo-B were validated.
We identify Nogo-C as a tumor suppressor gene in HCC and B-raf as a novel interacting protein. These findings provide new directions for the mechanism research of Nogo family.
神经突生长抑制蛋白（Nogos）由三个主要成员组成一个家族，其特征是具有保守的RHD结构域。在所有成员中，Nogo-B被发现显著升高并在肝硬化中起重要作用，而Nogo-C是最短的一个，很少受到关注。本研究的目的是探讨Nogo-C与肝细胞癌（HCC）的相关性及机制。
用定量RT-PCR检测配对的HCC标本中Nogo-C的表达。通过细胞增殖试验和集落形成试验评估Nogo-C在SMMC-7721和WRL-68 HCC细胞系中过表达的功能。使用亲和纯化结合高灵敏度质谱技术对Nogo-C结合蛋白进行全蛋白质组鉴定。使用免疫共沉淀和免疫荧光共聚焦试验确认蛋白质相互作用。
与相邻的病理正常组织相比，HCC标本中Nogo-C mRNA的表达极度下调，且与更大的肿瘤大小和更差的预后显著相关。Nogo-C在HCC细胞系中的过表达导致细胞生长受到抑制。共检测到73种与Nogo-C相关的蛋白质，其中B-raf和Nogo-B得到验证。
我们确定Nogo-C为HCC中的肿瘤抑制基因，B-raf为一种新的相互作用蛋白。这些发现为Nogo家族的机制研究提供了新的方向。
