Purpose

The etiology of nasopharyngeal carcinoma (NPC) is multifactorial, complex and not fully characterized yet. MicroRNAs (miRNAs or miRs) have been found to contribute to the development and progression of NPC. Here, we aimed to investigate the putative role of miR-129-5p in NPC lymphangiogenesis and lymph node metastasis (LNM), including the involvement of its target gene ZIC2 and the Hedgehog signaling pathway.

Methods

The expression of miR-129-5p and ZIC2 in primary NPC tissues was assessed using RT-qPCR and Western blot analyses, followed by LNM and lymph vessel density (LVD) correlation analyses. A direct interaction between miR-129-5p and ZIC2 was verified using a dual-luciferase reporter assay. Gain- and loss-of-function experiments were conducted to investigate the effects of miR-129-5p and ZIC2 expression on NPC cell invasion, migration and proliferation in vitro, as well as on LDV and LNM in nude mice in vivo. Additionally, RT-qPCR and Western blot analyses were performed to determine the expression levels of Hedgehog signaling pathway-related factors.

Results

We found that ZIC2 was highly expressed, and miR-129-5p was lowly expressed, in primary NPC tissues. In addition, we found that miR-129-5p can directly bind to and reduce ZIC2 expression. LVD was found to be negatively correlated with miR-129-5p and to be positively correlated with ZIC2 expression. Concomitantly, we found that miR-129-5p abrogated activation of the Hedgehog signaling pathway via ZIC2 targeting, leading to suppression of NPC cell invasion, migration and proliferation in vitro as well as suppression of LNM and LVD in vivo.

Conclusions

From our data we conclude that miR-129-5p, by decreasing ZIC2 expression, may inhibit NPC lymphangiogenesis and LNM through suppression of the Hedgehog signaling pathway.

中文翻译：

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MicroRNA-129-5p通过靶向ZIC2抑制鼻咽癌淋巴管生成和淋巴结转移

目的

鼻咽癌（NPC）的病因是多因素的，复杂的并且尚未完全表征。已经发现MicroRNA（miRNA或miR）有助于NPC的发展和进程。在这里，我们旨在调查miR-129-5p在NPC淋巴管生成和淋巴结转移（LNM）中的假定作用，包括其靶基因ZIC2和Hedgehog信号通路的参与。

方法

使用RT-qPCR和Western blot分析评估miR-129-5p和ZIC2在原发NPC组织中的表达，然后进行LNM和淋巴管密度（LVD）相关分析。使用双重萤光素酶报告基因分析验证了miR-129-5p与ZIC2之间的直接相互作用。进行功能获得和丧失功能实验以研究miR-129-5p和ZIC2表达对体外NPC细胞侵袭，迁移和增殖以及体内裸鼠LDV和LNM的影响。另外，进行RT-qPCR和蛋白质印迹分析以确定刺猬信号通路相关因子的表达水平。

结果

我们发现ZIC2在原发性NPC组织中高表达，而miR-129-5p低表达。此外，我们发现miR-129-5p可以直接结合并减少ZIC2表达。发现LVD与miR-129-5p负相关，并与ZIC2表达正相关。同时，我们发现miR-129-5p通过ZIC2靶向消除了Hedgehog信号通路的激活，从而抑制了NPC细胞的体外侵袭，迁移和增殖以及体内对LNM和LVD的抑制。

结论

根据我们的数据，我们得出的结论是，miR-129-5p通过降低ZIC2表达，可以通过抑制Hedgehog信号通路来抑制NPC淋巴管生成和LNM。