长链非编码RNA核富集转录体1促进人肝癌细胞增殖和侵袭的研究  

Long noncoding RNA nuclear enriched abundant transcript 1 promotes proliferation and invasion of hepatocellular carcinoma cells

作　　者：莽源祎 李立[1] 冉江华[1] 张升宁[1] 刘静[1] 李来邦[1] 陈奕明[1] 刘剑[1] 高杨[1] 任刚[1] 

机构地区：[1]昆明医科大学附属甘美医院(昆明市第一人民医院)肝胆胰血管外科,650011

出　　处：《中华实验外科杂志》2016年第10期2299-2301,共3页Chinese Journal of Experimental Surgery

基　　金：昆明市科技计划项目（2012-02-03-A-H-04-0001）

摘　　要：目的 在人肝细胞癌（HCC）标本中检测癌和癌旁组织中长链非编码RNA（lncRNA）核富集转录体1（NEAT1）的表达差异,应用化学合成小干扰RNA （siRNA）抑制两组人肝癌细胞株（HepG2和SMMC-7721）NEAT1的转录,观察NEAT1转录降低后对HCC细胞的表达谱、侵袭和增殖功能的影响.方法 利用Trizol-氯仿提取HCC组织标本和癌旁组织（n=12）,细胞株（HepG2、SMMC-772、HCC-LM3）中的总RNA,实时定量反转录聚合酶链反应（RT-qPCR）检测各组NEAT1表达差异[甘油醛-3-磷酸脱氢酶（GAPDH）作为内参],使用转染试剂Lipofectamine 2000和siRNANEAT1（终质量浓度100nmol/L）对HepG2和SMMC-7721细胞进行NEAT1敲低,RT-qPCR检测NEAT1敲低效果,并对NEAT1敲低组（NEAT1表达量＜对照组40％）和对照组细胞（n=2）进行表达谱测序,检测NEAT1敲低后对HCC细胞各肿瘤相关基因表达的影响,对NEAT1敲低组和对照组细胞进行羧基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）标记（终质量浓度5μmol/L）的细胞（2×10^5个/孔）增殖检测、细胞计数试剂盒（CCK-8）进行细胞增殖检测（10 μl/2 000个细胞）、Transwell细胞侵袭移动能力检测来探究NEAT1对HCC细胞（2×10^5个/孔）增殖和侵袭功能的影响.结果 NEAT1在HCC细胞和癌组织中表达量高于癌旁组织（P＜0.01）,敲低HCC细胞株NEAT1表达导致很多参与肿瘤发生、发展通路的基因表达量改变（229个基因表达上调,148个基因表达下调,表达量改变超过2倍且P＜0.05）,敲低HCC细胞株NEAT1表达降低了HCC细胞体外增殖（P＜0.01）、侵袭（P＜0.01）的能力.结论 lncRNA NEAT1在人肝细胞癌的发生、发展中发挥了原癌基因的作用.Objective To detect the expression level of nuclear enriched abundant transcript 1（NEAT1） in hepatocellular carcinoma （HCC） cells and to determine the roles,and functional mechanisms of NEAT1 in the proliferation and invasion of HCC cells using small interfering RNA （siRNA） against NEAT1.Methods Total RNA was extracted from tissues and cell lines using Trizol-Chloroform reagent （n =12）.The expression of each RNA was detected by real-time quantitative reverse transcriptase-polymerase chain reaction （RT-qPCR） and normalized to that of glyceraldehyde-3-phosphate dehydrogenase （GAPDH）.HCC cell lines HepG2 and SMMC-7721 were transfected using siRNA NEAT1 （100 nmol/L） and Lipofectamine 2000.The effect of NEAT1-knockdown was detected by RT-qPCR.To focus on the function and potential molecular mechanism of NEAT1 in HCC,we profiled its gene expression pattern by gene sequencing and detected proliferation and invasion in NEAT1-knockdown HCC cell lines （n =2）.Results It was found that NEAT1 was up-regulated in HCC tissues and cell lines （P 〈0.01）.Knockdown of NEAT1 altered global gene expression patterns in HCC cells （229 genes up-regulated,and 148 genes down-regulated,fold change 〉 2,P 〈 0.05）.NEAT1-knockdown inhibited cell proliferation （P 〈 0.01）,migration and invasion （P 〈 0.01） in HCC cell lines.Conclusion Long noncoding RNA NEAT1 as an oncogene played an important role in HCC progression.

关 键 词：癌  肝细胞 长链非编码RNA 核富集转录体1 增殖 侵袭 

分 类 号：R735.7[医药卫生—肿瘤]