Application of Ninjurin, a novel adhesion molecule, to diagnosis and therapeutics of primary hepatocellular carcinoma

Ninjurin是一种新的蛋白质，在神经损伤后上调。据报道，Ninjurin表现出嗜同性粘附分子的性质，并在神经突起再生中起重要作用。我们发现，在大鼠肝再生模型中，Ninjurin的表达在增殖期后的分化期增强。此外，Ninjurin在人肝癌组织的癌灶中表达降低。为了确定Ninjurin在人肝细胞中表达的生物学后果，我们建立了组成型过表达Ninjurin蛋白的Huh 7，并进行了以下检查：（1）用血细胞计数器分析生长曲线，（2）流式细胞术分析细胞周期，（3）Western blot分析细胞周期相关蛋白。与对照细胞相比，过表达Ninjurin的Huh 7细胞的增殖受到抑制。细胞周期在G1/S期阻滞，S期细胞比例从45%下降到25%。Ninjurin诱导的细胞凋亡阻滞伴随着p21表达水平的升高和Cdk 2、Cdk 4、Cdk 6表达水平的降低。而Cdk抑制因子p16、p27、cyclin D1、Cyclin E的表达无明显变化。相反，Cyclin A的表达略有下降。以这种方式，Ninjurin诱导细胞周期激活剂的下调和细胞周期抑制剂的上调，随后抑制细胞生长。这些观察结果表明，Ninjurin在HCC中的低表达可能有助于肝癌细胞的生长。因此，Ninjurin的诱导有望抑制肝癌的生长，这可能为肝癌的治疗提供一种新的策略。

Ninjurin is a novel protein that is up regulated after nerve injury. It is reported that Ninjurin demonstrates properties of a homophilic adhesion molecule and plays an important role in neurite regeneration. We found that, in rat liver regeneration model, Ninjurin expression was enhanced in the differentiation phase after proliferation phase. In addition, expression of Ninjurin was decreased in cancerous lesion of human hepatocellular carcinoma tissue. To determine the biological consequence of Ninjurin expression in human hepatocytes, we established Huh7 constitutively over-expressing Ninjurin protein and performed the following examinations, (1) analysis of growth curve with hemocytometer, (2) flow cytometric analysis of cell cycle, (3) Western blot analysis of cell cycle related protein. Proliferation of Huh7 cells over-expressing Ninjurin was inhibited compared to that of control cells. Cell cycle was arrested at G1/S checkpoints on a fluorescence-activated cell sorter, and the fraction of S phase declined from 45% to 25%. The arrest induced by Ninjurin was accompanied by the accumulation of p21 expression level and decreases of Cdk2, Cdk4, Cdk6 expression levels. But there is no change in the expression of Cdk inhibitor p16, p27, cyclin D1, and Cyclin E expression. In contrast, expression of Cyclin A was slightly decreased. In this way, Ninjurin induces both down-regulation of cell cycle activators and up-regulation of cell cycle inhibitor, followed by inhibition of cell growth. These observations suggest that the low expression of Ninjurin in HCC may contribute to growth of hepatoma-cells. Therefore, induction of Ninjurin can be expected to suppress growth of hepatoma, which may provide a novel strategy for hepatocellular carcinoma.