Naked1通过调控P53抑制肝癌细胞增殖的机制研究

It has been reported that Naked1(NKD1) was abnormal expressed in hepatoblastoma and other tumors, but the role of NKD1 on hepatocellular carcinoma (HCC) cell proliferation still needs to be further addressed. Our previous study has found that interference the expression of NKD1 protein promoted cell proliferation of HCC and decreased the level of P53 protein, meanwhile, the level of P21 protein which was associated with cell prolifetation was also decreased. The results of IHC also showed that NKD1 protein is negatively associated with hepatic tumor size. Based on the above results and combined with the existing literatures, we suppose that NKD1 maybe inhibits HCC cell proliferation via P53 protein. In order to verify this hypothesis, we suppose to detect NKD1 and P53 expression pattern in HCC tissues and analyze their relationship with HCC clinical pathologic factors and patients’ prognosis. We will next investigate the mechanism of NKD1 regulating hepatoblastoma cell proliferation via P53 through the methods of NKD1 over-expression in different human hepatoblastoma cell lines which contain mutant P53 and lack of P53, and then further confirm our guess through the nude mouse tumorigenicity assay in vitro. In this study, we study the mechanism of NKD1 on regulating HCC cell proliferation via P53 the first time. The goal of this study is expected to provide a theoretical basis for NKD1 as a new target of anti-HCC therapy.

目前已证实，Naked1（NKD1）在肝母细胞瘤和其它一些肿瘤中有异常表达，但NKD1对肝癌细胞增殖的调控及机制依然不清。我们前期预实验发现：在肝癌细胞中干扰NKD1表达能促进肝癌细胞增殖；并明显下调P53及其下游与增殖密切相关的P21蛋白水平；且免疫组化结果也表明NKD1的蛋白表达与肝癌肿瘤大小呈现负相关。基于以上结果及已有文献报道，我们提出假说：NKD1可能通过调控P53蛋白表达而抑制肝癌细胞增殖。为了验证此假说，本项目拟在肝癌组织中检测NKD1、P53表达，分析两者的相关性及NKD1表达与肝癌临床病理特征及患者预后关系；在含有P53野生型和缺失型的不同肝癌细胞株中“过表达”NKD1，研究细胞组织情况，并探讨NKD1调控P53蛋白的具体机制；最后通过裸鼠移植瘤实验进一步验证。本项目首次阐述NKD1通过P53蛋白影响肝癌细胞增殖及机制，为临床以NKD1作为肝癌治疗的新靶点提供理论依据。

目前已证实，Naked1（NKD1）在肝母细胞瘤和其它一些肿瘤中有异常表达，但NKD1对肝癌细胞生物学功能的调控及机制依然不清。我们从69例肝癌和配对的癌旁正常肝组织中提取蛋白，通过Western Blot的实验方法检测NKD1的蛋白表达情况，结果首次发现：NKD1蛋白在肝癌组织中的表达水平明显低于癌旁正常肝组织。另外，我们在58例具有完整的病例资料的石蜡标本中，通过免疫组织化学技术的方法，检测NKD1的表达定位情况，结果发现NKD1蛋白主要定位于肝癌细胞浆中，统计分析首次发现NKD1的低表达与肝癌患者的不良分化、肝内外转移、肿瘤大小和不良预后密切相关。.同时，我们在肝癌细胞株中构建了“过表达”和“沉默”NKD1的稳定转染细胞株，功能学实验发现，NKD1能够改变肝癌细胞的增殖和凋亡能力。体内裸鼠皮下成瘤实验也证实了NKD1蛋白与肝癌的肿瘤大小密切相关。机制探讨发现，NKD1通过诱导p53的蛋白表达后进而抑制了肝癌细胞的增殖能力。.最后，在构建稳定转染的肝癌细胞株的基础上，通过体内外实验研究发现，NKD1蛋白负向调控肝癌细胞的侵袭转移能力。机制研究发现，NKD1可以在细胞浆中与Rac1结合，并促进Rac1的范素化降解。而Rac1可以反过来通过调控EZH2的蛋白水平激活NKD1的基因转录，从而形成了一个负反馈的调节环。正常肝细胞中存有这一平衡体系，然而一旦这种平衡被打破，就使得肝癌得以发生发展。.本项目成果对阐明肝癌发生发展的分子机制和挖掘潜在治疗策略提供重要的理论依据，NKD1可能成为肝癌复发转移潜在的治疗靶点。