Hsa-miR-486-5p调控CXCL13在PI3K/Akt介导喉鳞癌EMT及侵袭转移中的机制研究

Invasion and metastasis is major risk factors of recurrence and death in laryngeal squamous carcinoma (LSCC). Although PI3K/Akt pathway, which could be activated by chemokine CXCL13, played an important role in tumorous invasion and metastasis, especially in epithelial-mesenchymal transition (EMT), the mechanism of regulation has been unknown. We have got particular microRNA-mRNA negative correlation profile in LSCC with lymph node metastasis by use of mRNA and microRNA array. We found that genes affiliated to this profile were significantly enriched in migration, invasion and malignant proliferation through bioinformatics analysis. We observed that CXCL13 was significantly increased, but Has-miR-486-5p down-regulated significantly which could combine with 3’-UTR region and degrade CXCL13 by our experiment of sensor report system. We speculate the regulation between Hsa-miR-486-5p and CXCL13 might be essential in EMT procession mediated by PI3K/Akt. In this study, we will illuminate the mechanism of regulation between Hsa-miR-486-5p and CXCL13 in EMT, invasion and metastasis mediated by PI3K/Akt, and absorbingly mechanism of transcription suppression of Hsa-miR-486-5p caused by methylation. Meanwhile, the feature of clinicopathological parameters and prognosis in different LSCC patients, classified with molecular, will be analyzed in order to summarize their clinical significance and provide new targets for the treatment in LSCC.

侵袭转移是喉鳞癌患者复发及死亡主要危险因素。有研究发现趋化因子CXCL13可激活PI3K/Akt通路并促进癌细胞间质转化(EMT)及侵袭转移,但调控机制不明。我们前期获得转移喉鳞癌microRNA/mRNA联合表达谱表明上调基因在细胞迁移/侵袭等中显著富集，其中CXCL13及PI3K/Akt关键分子显著上调，而miR-486-5p显著下调并首次鉴定CXCL13为其靶基因。申请人推测miR-486-5p通过调控CXCL13在PI3K/Akt介导喉鳞癌EMT及侵袭转移中发挥重要作用。本研究拟从体内外层面探讨miR-486-5p调控CXCL13对PI3K/Akt介导喉鳞癌EMT及侵袭转移的影响并证实甲基化可能是miR-486-5p转录受阻的重要机制;同时分析miR-486-5p及CXCL13表达与喉鳞癌临床病理参数及预后关系,并对患者分子分型。旨在进一步阐明喉鳞癌侵袭转移机制,为治疗提供新靶点。

喉鳞癌位居头颈部恶性肿瘤第二位，在包括山西在内的我国北方地区高发，患者一旦罹患喉鳞癌，对呼吸、发声及吞咽等重要生理功能产生严重损害。侵袭转移是喉鳞癌典型的恶性生物学特点之一，是喉鳞癌术后复发及不良预后的主要危险因素。GO及KEGG分析显示EMT中的重要通路PI3K/Akt其上的关键分子呈显著变化，如PIK3CA上调3.7倍，AKT2 上调2.0倍；EMT重要分子MMP-1上调59.4倍，MMP-11上调37.4倍。特别是位于激活PI3K/Akt通路上游的趋化因子CXCL13上调最显著（61倍），受体CXCR5上调1.9倍，而可靶向结合CXCL13 3’-UTR的miR-486-5p却下调6.1倍。本项目发现Hsa-miR-486-5p在喉鳞癌中低表达，与其靶向结合的CXCL13及受体CXCR5在喉鳞癌中高表达，同时验证了Hsa-miR-486-5p与CXCL13直接结合的靶向关系。通过甲基化检测发现Hsa-miR-486-5p启动子高甲基化可能是其在喉鳞癌中转录受阻的重要表观调控机制。体外过表达Hsa-miR-486-5p后，可明显抑制喉鳞癌细胞系Hep-2、TU177的迁移及侵袭能力。敲减CXCL13后，喉鳞癌细胞系Hep-2、TU177的迁移及侵袭能力亦受到遏制，过表达CXCL13则可恢复喉鳞癌细胞系Hep-2、TU177的迁移及侵袭的恶性能力。Hsa-miR-486-5p/CXCL13可通过改变EMT关键分子，从而影响喉鳞癌细胞系的迁移及侵袭能力，在这一生物学过程中，PI3K/AKT信号通路的激活是重要的介导机制之一。恢复Hsa-miR-486-5p或敲减CXCL13，均可抑制Hep-2、TU177的裸鼠移体内成瘤能力。趋化因子CXCL13 及其受体蛋白 CXCR5与喉鳞癌TNM分期、淋巴结转移及临床分期呈显著相关性，特别是喉鳞癌血清中CXCL13水平显著高于正常对照人群，可能是喉鳞癌预后评估新的重要潜力因子。