长链非编码RNA HNF1A-AS1在喉癌淋巴结转移中的作用机制及临床意义

In our previous study,we found that the differential expression of HNF1A-AS1 was most significant between laryngeal cancer tissues and matched cervical lymph node metastasis through high-throughput screening with lcnRNA microarray, and the preliminary experimental results showed that disordered expression of HNF1A-AS1 could result in morphological changes of Hep-2 cells and affect invasion ability of cancer cells, indicating that HNF1A-AS1 may be involved in participating in lymph node metastasis of laryngeal cancer. However, there is no relevant reports about the action mechanism and clinical implication of HNF1A-AS1 in lymph node metastasis of laryngeal cancer. Therefore, this project is a continuation of the previous studies, in which we aim to confirm the possible mechanism that HNF1A-AS1 could regulate snail1 and induce epithelial-interstitial transformation to promote lymph node metastasis of laryngeal cancer after analyzing the effects of disordered expression of HNF1A-AS1 on metastasis of laryngeal cancer cells, providing new theoretical basis for lymph node metastasis of laryngeal cancer. Furthermore, we want to explore the possibility of HNF1A-AS1 served as a molecular marker for lymph node metastasis of laryngeal cancer by simultaneous analysis of the relationship between HNF1A-AS1 expression and indicators such as clinical stages, prognosis and lymph node metastasis status, ultimately providing a reference for research about clinical implication of HNF1A-AS1 in lymph node metastasis of laryngeal cancer.

在前期研究中，我们通过lcnRNA芯片高通量筛选喉癌组织和配对颈部转移淋巴结差异表达的lncRNA，结果发现HNF1A-AS1差异表达最为显著，且预实验结果显示HNF1A-AS1表达失调可引起喉癌Hep-2细胞形态改变，影响癌细胞的侵袭能力，提示HNF1A-AS1参与喉癌淋巴结转移。但其作用机制是什么？有何临床意义？目前还没有相关的研究报道。因此，本项目是前期研究的继续和深入，拟检测HNF1A-AS1表达失调对喉癌细胞转移能力的影响，试图证实HNF1A-AS1通过调控Snail1诱导上皮-间质转化促进喉癌淋巴结转移的可能机制，为喉癌淋巴结转移提供新的理论依据；通过同步分析喉癌组织和患者血清中HNF1A-AS1的表达同喉癌临床分期、预后及淋巴结转移状态等指标之间的相关性，探索HNF1A-AS1作为喉癌淋巴结转移分子标记物的可能性，为研究HNF1A-AS1在喉癌淋巴结转移的临床意义提供依据。

喉癌淋巴结转移的分子机制目前仍不明确。在前期研究中，我们通过lcnRNA芯片高通量筛选喉癌组织和配对颈部转移淋巴结差异表达的lncRNA，结果发现HNF1A-AS1差异表达最为显著，且初步实验结果显示HNF1A-AS1表达失调可引起喉癌Hep-2细胞形态改变，影响癌细胞的侵袭能力，提示HNF1A-AS1参与喉癌淋巴结转移。在此基础上，本项目以喉癌组织、癌旁对照组织和配对的转移淋巴结为研究对象，研究HNF1A-AS1的表达水平与喉癌淋巴结转移状态等临床病理学参数的相关性，结果发现转移淋巴结HNF1A-AS1的表达水平显著低于喉癌组织和癌旁对照组织，且与喉癌淋巴结转移状态等显著相关；其次，以Hep-2和Fadu细胞为研究对象，分别干扰HNF1A-AS1和过表达HNF1A-AS1，应用CCK-8检测、流式细胞凋亡检测、细胞迁移实验、transwell侵袭实验、细胞粘附实验、明胶酶谱实验、细胞骨架蛋白染色等实验方法探索HNF1A-AS1表达失调对喉癌细胞增殖、迁移、侵袭等生物学行为的影响，结果发现Hep-2细胞干扰HNF1A-AS1后细胞迁移和侵袭能力显著增强，Fadu细胞过表达HNF1A-AS1后细胞迁移和侵袭能力显著减弱，且与MMP-2、MMP-9等细胞外基质金属蛋白酶和细胞骨架蛋白有关。最后，应用western blot和免疫荧光法检测HNF1A-AS1表达失调对EMT标志物表达的影响，结果发现干扰Hep-2细胞HNF1A-AS1的表达后细胞形态由圆钝形演变为长梭形，E-cadherin表达水平显著下降，而Snail1、Slug、Vimentin、N-cadherin等间质标志物表达水平显著上调，Fadu细胞过表达HNF1A-AS1后细胞形态改变与EMT标志物表达变化趋势正好相反，说明HNF1A-AS1的作用与调控EMT有关。总之，本项目探索HNF1A-AS1在喉癌淋巴结转移中的作用，阐明HNF1A-AS1通过调控EMT参与喉癌淋巴结转移的具体分子机制，为进一步理解喉癌淋巴结转移的分子机制提供科学依据，也为开发阻断喉癌淋巴结转移的靶向药物提供理论基础。