喉鳞状细胞癌进展中FOXP4和miR-4476协同调控表达的临床意义  

Clinical significance of FOXP4 and mi R-4476 regulating expressions in progression of laryngeal squamous cell carcinoma

作　　者：赵岩[1] 刘胜辉[1] 王晶田[1] 石艳凤 石健[1] 吴干勋[1] 兰利利[1] ZHAO Yan;LIU Shenghui;WANG Jingtian;SHI Yanfeng;SHI Jian;WU Ganxun;LAN Lili(Department of Otolaryngology,Fourth Hospital of Hebei Medical University,Shijiazhuang 050011,Hebei Province,China;Department of Ophthalmology and Otorhinolar yngology,County Hospital of Luannan,Tangshan 063500,Hebei Province,China)

机构地区：[1]河北医科大学第四医院耳鼻喉头颈外科,河北石家庄050011 [2]河北省滦南县医院五官科,河北唐山063500

出　　处：《肿瘤》2020年第9期602-613,共12页Tumor

基　　金：河北省医学科学研究重点课题(编号:20201511;20180588)。

摘　　要：目的:检测微RNA(microRNA,miRNA,miR)-4476在喉鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)组织及人喉癌细胞系中的表达水平,以及其对喉癌细胞体外增殖、迁移及侵袭能力的影响,并检测miR-4476与转录因子叉头框蛋白4(fork head box protein 4,FOXP4)的相关性及可能的结合位点。方法:应用实时荧光定量PCR法和免疫组织化学SP法检测LSCC组织及其相应癌旁正常组织中FOXP4 mRNA和蛋白的表达情况。应用在线网站预测与FOXP4有调控作用的miRNA,采用实时荧光定量PCR法检测miR-4476在LSCC组织与其相应癌旁正常组织及4种人喉癌细胞系中的表达情况,并检测其与FOXP4表达的相关性。采用染色体免疫共沉淀技术检测FOXP4与miR-4476启动子区的结合情况。将pcDNA3.1-FOXP4与报告基因载体pGL3-miR-4476启动子区共转染至293T细胞中,通过检测荧光素酶报告基因荧光信号活性分析FOXP4对miR-4476启动子区的影响。将miR-4476模拟物及抑制物分别转染人喉癌TU177和AMC-HN-8细胞,用实时荧光定量PCR法检测转染效率,然后采用MTS实验、细胞克隆形成实验、Transwell小室迁移及侵袭实验分别检测miR-4476表达调控对喉癌细胞增殖、迁移及侵袭的影响。结果:在LSCC组织中FOXP4 mRNA的表达水平(P<0.05)及蛋白阳性表达率(P<0.01)均高于正常组织,且蛋白表达阳性率与肿瘤的淋巴结转移和TNM分期相关(P值均<0.05)。LSCC组织中miR-4476表达水平明显高于癌旁正常组织(P<0.01),与肿瘤的TNM分期及淋巴结转移相关(P值均<0.05)。LSCC组织中miR-4476与FOXP4的表达呈正相关(R=0.455,P<0.01),而且FOXP4与miR-4476的启动子区结合(P<0.01),可能具有促进miR-4476启动子活性的作用(P<0.01)。喉癌细胞中miR-4476表达均高于对照组(P<0.05),转染miR-4476模拟物后TU177细胞中miR-4476表达水平明显升高(P<0.01),而转染miR-4476抑制物后AMC-HN-8细胞中miR-4476表达水平明显降低(P<0.05)。与对照组相比,转染miObjective:To detect the expression of microRNA(miRNA,miR)-4476 in laryngeal squamous cell carcinoma(LSCC)tissues and human laryngeal cancer cell lines,and its effects on the proliferation,migration,and invasion of laryngeal cancer cells in vitro.To explore the correlation of miR-4476 with fork head box protein 4(FOXP4)and the possible binding site.Methods:Real-time fluorescent quantitative PCR and immunohistochemistry SP method were used to detect the FOXP4 mRNA and protein expression in LSCC tissues and the corresponding normal tissues.The online websites were used to predict the miRNAs with regulatory relationship of FOXP4.The expression of miR-4476 in LSCC tissues and their adjacent normal tissues and human laryngeal cancer cell lines was detected by real-time fluorescent quantitative PCR,and the correlation between miR-4476 and FOXP4 expression was analyzed.Chromatin immunoprecipitation assay was used to detect the binding of FOXP4 to the miR-4476 promoter region.pcDNA3.1-FOXP4 was co-transfected with the reporter gene vector pGL3-miR-4476 promoter region into 293T cells,and the effect of FOXP4 on the miR-4476 promoter region was analyzed by detecting the luciferase reporter gene fluorescence signal activity.miR-4476 mimics and miR-4476 inhibitor were used to transfect into laryngeal cancer cells,and the transfection efficiency was measured by real-time fluorescent quantitative PCR.The effects of miR-4476 on the proliferation,migration and invasion of laryngeal cancer cells were tested by MTS assay,clone formation assay,Transwell chamber migration and invasion assays,respectively.Results:In LSCC tissues,the mRNA expression(P<0.05)and protein positive expression rate(P<0.01)of FOXP4 were higher than those in normal adjacent tissues,the protein positive expression rate was related to lymph node metastasis and TNM stage(both P<0.05);the expression of miR-4476 was significantly higher than that in normal adjacent tissues(P<0.01),which was related to the TNM stage of tumors and lymph node metastasis(both P<0.05).I

关 键 词：喉肿瘤 微RNAS 叉头转录因子类 miR-4476 FOXP4 生物学行为 

分 类 号：R739.65[医药卫生—肿瘤]