微小RNA-519b-3p对人喉癌Hep-2细胞增殖的抑制作用及其机制  

Growth inhibitory effect of microRNA-519b-3p on larynx squamous Hep-2 cells

作　　者：沈志森[1] 詹国文[1] 邓红霞[1] 康骋[1] 郭俊明 

机构地区：[1]浙江省宁波市医疗中心李惠利医院耳鼻咽喉头颈外科,315040 [2]浙江省病理生理学技术研究重点实验室

出　　处：《中华耳鼻咽喉头颈外科杂志》2014年第2期151-156,共6页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：浙江省医药卫生科技计划项目（2012ZDA042）;宁波市重大择优委托项目（2012C5015）;宁波市科技创新团队（2012B82019）;宁波市自然科学基金（2012A610208;2012A610217）

摘　　要：目的 探讨微小RNA-519b-3p（miR-519b-3p）对人喉癌Hep-2细胞增殖的影响及其在喉癌发生发展过程中的作用及机制.方法 通过四甲基偶氮唑蓝（MTT）和流式细胞仪分别检测转染miR-519b-3p类似物前后Hep-2细胞生长和细胞周期的变化,采用反转录聚合酶链反应（RT-PCR）和Western blot分别在mRNA和蛋白水平检测细胞中相关基因和蛋白的表达.实时定量RT-PCR技术检测48对喉癌组织及癌旁组织中miR-519b-3p的表达水平.结果 喉癌组织中miR-519b-3p的表达水平明显低于癌旁组织（S△Ct=2.989,f =2.693,P＜0.01）.miR-519b-3p类似物能显著抑制Hep-2细胞的增殖,使细胞周期被阻滞在G2/M期（10.29％±4.63％,（x）±s,t=4.395,P＜0.05）,并明显减少S期细胞（7.56％±2.05％,t=3.555,P＜0.05）,使细胞周期蛋白依赖性激酶1（cyclin dependent kinase 1,CDKl）表达显著增加,而显著抑制CDK2和细胞周期蛋白A（Cyclin A）的表达.RT-PCR和Western blot结果显示,miR-519b-3p类似物在不影响HuR和环氧化酶-2（cyclooxgenase-2,COX-2）mRNA表达水平情况下,却明显下调其蛋白表达水平.结论 miR-519b-3p在喉癌组织中低表达,它可能作为抑癌因子通过翻译后抑制HuR和COX-2的表达而使细胞周期被阻滞在G2/M期,从而抑制细胞生长.Objective To investigate the effects of microRNA-519b-3p （miR-519b-3p） on laryngeal carcinoma Hep-2 cell growth,and to analyze the underlying molecular mechanisms.Methods The effects of miR-519b-3p on the growth and cell cycle of Hep-2 cells transfected with miR-519b-3p mimic were tested by MTT assay and flow cytometry,respectively.The mRNA and protein expressions of the related genes were tested by reverse transcription polymerase chain reaction （RT-PCR） and Western blot,respectively.The expressions of miR-519b-3p were tested by real-time RT-PCR in 48 pairs of laryngeal carcinoma and adjacent tissue samples.Results The expression of miR-519b-3p in laryngeal carcinoma tissues was significant lower than that in adjacent non-cancerous tissues （SAct =2.989,t =2.693,P ＜ 0.01）.Increasing the level of miR-519b-3p inhibited significantly Hep-2 cell proliferation,arrested the cell cycle in the G2/M phase （10.29％ ± 4.63％,t =4.395,P ＜ O.05）,and decreased significantly the percentage of cells in the S phase （7.56％-± 2.05％,t =3.555,P ＜ O.05）,with the increase in the expression of cyclin dependent kinase （CDK） 1 and the decrease in the expressions of CDK 2 and Cyclin A.RT-PCR and Western blot showed that miR-519b-3p down-regulated the protein but not mRNA expressions of HuR and cyclooxgenase-2 （COX-2） genes.Conclusions The expression of MiR-519b-3p as carcinoma suppressor gene is low in laryngeal carcinoma.The cell cycle of Hep-2 cells was arrested in the G2/M phase by MiR-519b-3p.

关 键 词：喉肿瘤 癌 鳞状细胞 微RNAS 细胞周期蛋白A 细胞系 肿瘤 细胞增殖 

分 类 号：R739.65[医药卫生—肿瘤]