NgR3通过上皮间质转化促进鼻咽癌细胞侵袭转移的机制研究

Distant metastasis remains the major reason of treatment failure for nasopharyngeal carcinoma (NPC) patients, but its molecular mechanism is still unknown, lacking of effective therapeutic target. Receptor membrane proteins, which are closely related to distant metastasis, may be considered as potential therapeutic targets for NPC. By using RNA deep-sequencing, genes of different expression had been screened in NPC specimens and normal nasopharyngeal specimens, and it was found that expression of Nogo-66 receptor related protein 3 (NgR3) was unregulated in NPC specimens. Further Immunohistochemical assay and prognostic analysis demonstrated that high expression of NgR3 might contribute to distant metastasis and poor prognosis. In vitro, expression of NgR3 was low in NPC cells of low metastatic ability, in which overexpression of NgR3 could downregulate the expression of E-cadherin, and enhance cell invasion and metastasis potency; whereas, knockdown of NgR3 could decrease migration and invasion ability. All these results indicate that NgR3 may be a potential therapeutic target for distant metastasis of NPC. However, there are few studies on NgR3 in malignant tumors, and the molecular mechanism is still elusive. In this project, the CRISPR knockout, distant metastasis model in nude mice, Co-IP and mass spectrometry could be used to study the function and mechanism of NgR3 and its ligands in NPC metastasis, hereby providing a new insight into the effective therapeutic target of NPC.

鼻咽癌治疗失败的主要原因是远处转移，但其分子机制不明，并缺乏有效治疗靶标。与鼻咽癌远处转移密切相关的受体膜蛋白是潜在治疗靶点。本项目前期应用RNA深度测序技术在鼻咽癌组织和鼻咽正常组织mRNA中筛选差异表达基因，发现Nogo-66受体相关蛋白3（NgR3）在鼻咽癌组织中高表达；免疫组化和预后分析显示NgR3高表达患者更容易发生远处转移，而且预后更差；体外实验发现NgR3在鼻咽癌低转移细胞株中低表达，过表达NgR3可下调E-cadherin表达，且细胞迁移、侵袭能力增强；敲除NgR3后细胞迁移、侵袭能力减弱，这些结果提示NgR3可能成为鼻咽癌远处转移的潜在治疗靶点。然而，目前关于NgR3在恶性肿瘤中的作用研究较少，机制不清。本课题拟通过CRISPR基因敲除技术、裸鼠转移模型、免疫共沉淀以及质谱技术等研究NgR3及配体在鼻咽癌远处转移中的作用及其分子机制，为寻找鼻咽癌转移有效治疗靶点提供依据。

鼻咽癌高发于我国华南地区，远处转移是治疗失败的主要原因，是鼻咽癌研究领域面临的难点问题和前沿热点。本课题组发现NgR3在鼻咽癌中高表达，且与鼻咽癌患者的不良预后相关。NgR3可促进鼻咽癌细胞的迁移、侵袭和肺转移，并发现NgR3通过FAK-src介导EMT，从而促进鼻咽癌的侵袭转移作用。进一步的研究发现NgR3的配体Nogo-B在鼻咽癌组织和细胞中高表达。免疫组化检测结合临床资料分析发现高表达Nogo-B的鼻咽癌更易发生淋巴结转移和远处转移，并与鼻咽癌预后不良相关。体内外实验证实过表达Nogo-B能增强鼻咽癌细胞的迁移、侵袭、转移能力。Co-IP和共定位均提示Nogo-B和NgR3有直接相互作用，这提示Nogo-B可能通过NgR3发挥侵袭转移作用。在过表达Nogo-B的鼻咽癌中，EMT相关蛋白和Rho蛋白表达上调，使用阻断剂CGG1423后，EMT相关蛋白表达下调，这提示Nogo-B可能通过EMT及Rho/SRG/MRTFA通路发挥侵袭转移作用。本研究明确了NgR3及其配体Nogo-B在鼻咽癌侵袭转移中的作用，为研究鼻咽癌侵袭转移的机制及靶向治疗提供新的思路。