转录因子FOXP4在喉鳞状细胞癌组织中的表达及其对喉鳞癌TU177细胞生物学特性的影响

Expression of transcription factor FOXP4 in laryngeal squamous cell carcinoma tissues and its effects on biological characteristics of laryngeal squamous cell carcinoma TU177 cells

目的:检测转录因子FOXP4(Forkhead box P4)在喉鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)组织和细胞系中的表达,及其对LSCC细胞TU177体外增殖、迁移、侵袭、细胞周期及凋亡的影响,并探讨其与上皮-间质转化(epithelialmesenchymal transition,EMT)进程间的关系。方法:从河北医科大学第四医院生物标本库选取2013年6月至2015年12月收治的40例LSCC手术患者的癌及癌旁组织标本,应用qPCR法检测FOXP4在LSCC与相应癌旁组织中的表达水平,应用qPCR法和Western blotting检测FOXP4在人LSCC细胞系(AMC-HN-8、TU177、TU686及TU212)中的表达水平。采用小干扰RNA敲低TU177细胞中FOXP4的表达,MTS实验、克隆形成实验、Transwell实验及流式细胞术分别检测FOXP4敲低对TU177细胞增殖、迁移、侵袭、周期及凋亡的影响。应用qPCR法检测si-FOXP4转染TU177细胞后EMT标志物N-钙黏蛋白(N-cad‐herin)、β-连环蛋白(β-catenin)、波形蛋白(Vimentin)、扭曲蛋白(Twist)、转录因子Snail及锌指E盒结合蛋白1(zine finger E box binding homeobox 1,ZEB1)mRNA的变化情况,应用Western blotting测定FOXP4敲低后N-cadherin、β-catenin、Vimentin及Twist蛋白水平的改变。结果:在LSCC组织中FOXP4的表达水平明显高于癌旁组织(P<0.05),且与肿瘤的TNM分期及淋巴结转移相关(均P<0.05)。FOXP4在LSCC细胞中的表达亦高于癌旁组织(P<0.05或P<0.01)。转染si-FOXP4的TU177细胞中FOXP4的表达显著低于对照组(P<0.01)。与对照组相比,敲低FOXP4可抑制TU177细胞的体外增殖、迁移和侵袭能力(均P<0.01),敲低FOXP4可使细胞周期阻滞于G0/G1期(P<0.01),减少S期的复制(P<0.01),促进细胞凋亡(P<0.01),敲低FOXP4可降低TU177细胞中N-cadherin、β-catenin、Vimentin、Twist、Snail、ZEB1的mRNA水平(P<0.05或P<0.01)以及N-cadherin、β-catenin、Vimentin、Twist的蛋白水平。结论:FOXP4的高表达可能与LSCC的发生和发展相关,FOXP4敲低可以抑制LSCC细胞的体外增殖、迁移与侵袭能力,使细胞G0/G1期阻滞及促进凋亡,且可能参与EMT进程。

Objective:To detect the expression of transcription factor FOXP4(Forkhead box P4)in laryngeal squamous cell carcinoma(LSCC)tissues and cell lines,and to investigate its effects on the proliferation,migration,invasion,cell cycle,and apoptosis of LSCC TU177 cells in vitro as well as to explore its relationship with epithelial-mesenchymal transition(EMT)process.Methods:A total of 40 pairs of tumor tissues and adjacent tissues that resected from LSCC patients were collected from the biological specimen bank of the Forth Hospital of Hebei Medical University between 2013 and 2015.The expression of FOXP4 in LSCC tissues and corresponding adjacent tissues was detected by qPCR.qPCR and Western blotting were used to detect the FOXP4 expression level in human LSCC cell lines(AMC-HN-8,TU177,TU686,and TU212).Small interfering RNA(si-RNA)was used to knock down FOXP4 expression in TU177 cells.The effects of FOXP4 knockdown on the proliferation,migration,invasion,cell cycle and apoptosis of TU177 cells were measured by MTS assay,clone formation assay,Transwell chamber migration and invasion assay,and flow cytometry,respectively.The mRNA levels of EMT markers N-cadherin,β-catenin,Vimentin,Twist,Snail and zine finger E box binding homeobox 1(ZEB1)after transfection of si-FOXP4 in TU177 cells were detected by qPCR.The changes of protein levels of N-cadherin,β-catenin,Vimentin and Twist after FOXP4 knockdown were measured by Western blotting.Results:The expression of FOXP4 in LSCC tissues was significantly higher than that in adjacent tissues(P<0.05),and it was related to the TNM stage of tumors and lymph node metastasis(all P<0.05).The expression of FOXP4 in LSCC cells was higher than that in the adjacent tissues(P<0.05 or P<0.01).The expression of FOXP4 in TU177 cells transfected with si-FOXP4 was significantly lower than that in the control group(P<0.01).Compared with the control group,knocking down FOXP4 could inhibit the proliferation,migration and invasion but promote the apoptosis of TU177 cells in vitro(all P<0.01),block the cell cycle at G0/G1 phase(P<0.01),and reduce cell replication in S phase(P<0.01);in addition,knocking down FOXP4 could reduce the mRNA levels of N-cadherin,β-catenin,Vimentin,Twist,Snail,ZEB1(P<0.05 or P<0.01)and the protein levels of N-cadherin,β-catenin,Vimentin,Twist in TU177 cells.Conclusion:The high expression of FOXP4 may be related to the occurrence and development of LSCC.FOXP4 knockdown can inhibit the proliferation,migration and invasion of laryngeal cancer cells in vitro,block cell cycle at G0/G1 phase,promote apoptosis,and may participate in the EMT process.