URG4在宫颈癌淋巴结转移中的作用及分子机制研究

Retroperitoneal lymph node metastasis is an important prognostic factor of cervical cancer. Up to now, and the mechanism of node metastasis remains unclear, and there is no molecular marker to predict node metastasis in cervical cancer. In our recent study, we found that URG4 was overexpressed in cervical cancer cell and tissue, and the expression level was an independent factor affecting prognosis and node metastasis. After URG4 gene was knocked down, the capacity of invasion and migration of cancer cell were significantly reduced. High-throughput sequencing revealed that URG4 was associated with Wnt/β-catenin signal pathway. Our further study showed that after silencing URG4 gene, the Wnt/β-catenin signal pathway and its downstream genes, which participate in the regulation of epithelial-mesenchymal transition (EMT), were inhibited. We speculated that URG4 promoted node metastasis in cervical cancer by regulating EMT through Wnt/β-catenin pathway. However, the action sites and detail mechanisms remain unclear and deserve further investigation. In this study, PCR arrays, shRNA silence, inhibitor interference and other techniques will be used to investigate the molecular mechanisms of URG4 regulates EMT through Wnt/β-catenin pathway in cellular models. Animal models will be used to verify the functions of URG4 on tumor metastasis in vivo. This study is aimed to illuminate the molecular mechanisms and clinical significance of URG4 promotes node metastasis in cervical cancer, and provide evidences for searching molecular markers predicting lymph node metastasis and novel therapeutic targets in cervical cancer.

腹膜后淋巴结转移是宫颈癌重要的预后影响因素，其发生机制尚不明确，也缺乏预测宫颈癌淋巴结转移的分子标记物。我们近期研究发现，URG4在宫颈癌中存在高表达，且表达水平是影响淋巴结转移和预后的独立因素；敲减URG4基因后，宫颈癌细胞的迁移、侵袭能力显著下降；高通量测序结果提示URG4与Wnt/β-catenin通路相关；沉默URG4表达后，Wnt/β-catenin通路及上皮细胞间质化（EMT）相关分子受到了抑制。我们推测URG4通过调控该通路引发EMT促进宫颈癌淋巴结转移，但具体作用位点和机制需要进一步研究。本项目拟在细胞模型中通过PCR芯片检测、shRNA沉默/过表达目标基因、抑制剂干预等方法明确URG4调控Wnt/β-catenin通路的分子机制，并用动物模型验证其功能。本项目将阐明URG4在宫颈癌淋巴结转移中的作用及分子机制，为寻找预测宫颈癌淋巴结转移的分子指标和治疗新靶点提供依据。

淋巴结转移是影响早期宫颈癌预后最重要的因素，目前，宫颈癌淋巴结转移发生的分子机制并未明确。我们的前期研究发现URG4是影响宫颈癌淋巴结转移和预后的独立因素；敲减URG4基因后，宫颈癌细胞的迁移、侵袭能力显著下降，我们推测URG4通过调控Wnt/β-catenin通路引发EMT促进宫颈癌淋巴结转移，但具体作用机制需要进一步研究。本项目开始后，我们对手术收集的新鲜癌、癌旁标本进行全转录组高通量测序，并结合前期敲减URG4后的测序数据进行生物学信息分析，发现一系列与URG4表达存在高度关联的基因，随后使用高内涵细胞功能筛选平台对这些基因功能进行筛选，最终确定长链非编码RNA LOC729737与RNA结合蛋白FASTKD2具有明显的功能表型，随后在宫颈癌细胞株中证实LOC729737与FASTKD2均能引起宫颈癌增殖、凋亡、侵袭、迁移能力的改变；在临床标本中，LOC729737与FASTKD2也存在高表达，且其表达水平与宫颈癌患者临床病理风险因素相关，是影响淋巴结转移和总体生存时间的独立因素；进一步分子生物学实验证实URG4通过LOC729737与FASTKD2调控Wnt/β-catenin活性，导致上皮细胞间质化，促进宫颈癌侵袭、转移。本研究发现对阐明宫颈癌淋巴结转移的分子机制有重要意义，有可能揭示宫颈癌新的检测和治疗靶点。