Abstract: Objective To investigate the effect and mechanism of FST on proliferation, invasion and migration ability of head and neck squamous cell carcinoma （HNSCC） cells. Methods The expression levels of FST in HNSCC cell lines HSC-3, Fadu, Tu686 and normal human oral epithelial cells HOK were determined by real-time quantitative PCR （qRT-PCR） and Western blot. FST knockdown cell lines were constructed using siRNA and shRNA. The effects of FST knockdown on proliferation, invasion and migration ability of HNSCC cells were analyzed by CCK8 assay, cell cloning formation assay, Transwell invasion and migration test and subcutaneous tumorigenesis test in nude mice. The total RNA of FST knockdown cell lines and the control group was sent to transcriptome sequencing analysis, and signal pathway enrichment analysis was performed for differentially expressed genes. Verification of possible signaling pathways was performed by qRT-PCR. The apoptosis of tumor cells was detected by flow cytometry and Western blot. Results The mRNA and protein expression levels of FST in Tu686, Fadu and HSC-3 were higher than those in HOK. After knockdown of FST, the number of tumor cells passing through the Transwell chamber decreased, the number of clones formed decreased, the proliferation rate of tumor cells slowed down and the subcutaneous tumor volume of nude mice were smaller compared with control group. Transcriptome sequencing results of FST knockdown tumor cells and control group showed that differentially expressed genes were significantly enriched in TGFβ signaling pathway. After FST knockdown, the proportion of apoptotic tumor cells was increased, and the expression of apoptosis-related protein Casepase3 was up-regulated. Conclusion After FST knockdown in HNSCC cells, tumor proliferation, invasion, migration ability and stemness were weakened, which may be related to TGFβ signaling pathway.

Key words: head and neck squamous carcinoma, follistatin, invasion, migration, proliferation

摘要： 目的 探讨卵泡抑素（FST）对头颈鳞癌细胞增殖、侵袭和迁移能力的影响及机制。方法 采用实时荧光定量PCR（qRT-PCR）和蛋白质印迹法检测头颈鳞癌细胞系HSC-3、Fadu、Tu686以及人正常人口腔上皮细胞HOK中FST的表达水平。利用siRNA和shRNA构建敲低FST的细胞株,通过CCK8、细胞克隆形成、Transwell侵袭迁移和裸鼠皮下成瘤实验,研究敲低FST对肿瘤细胞增殖、侵袭和迁移能力的影响;将敲低FST的细胞株和对照组的总RNA送检转录组测序,对差异表达的基因进行信号通路富集分析,利用qRT-PCR对TGFβ信号通路进行初步验证,通过Western blot和流式细胞技术检测肿瘤细胞凋亡情况。结果 FST在Tu686、Fadu和HSC-3细胞中mRNA及蛋白表达水平均高于HOK细胞。肿瘤细胞敲低FST后穿过Transwell小室的数目显著减少、形成的克隆数目显著减少、肿瘤细胞增殖速度变慢、裸鼠皮下成瘤体积变小。RNA测序结果显示,与对照组相比,敲低FST的肿瘤细胞差异表达的基因在TGFβ信号通路中有明显富集,敲低FST后肿瘤细胞凋亡比例增加,凋亡相关蛋白Casepase3表达上调。结论 头颈鳞癌肿瘤细胞敲低FST后肿瘤增殖、侵袭和迁移能力以及干性减弱,可能和TGFβ信号通路有关。

关键词: 头颈鳞癌, 卵泡抑素, 侵袭, 迁移, 增殖