沉默EMS1/cortactin基因对肝癌HepG2细胞迁移、侵袭和细胞周期的影响  

Effects of silencing EMS1/cortactin gene on migration,invasion and cell cycle of human hepatocellular carcinoma cell line HepG2

作　　者：周家名[1,2] 陈莉[1,2] 王桂兰[2] 秦婧[2] 吴圆圆[2] 

机构地区：[1]南通大学杏林学院医学部,江苏南通226001 [2]南通大学医学院病理系,江苏南通226001

出　　处：《基础医学与临床》2016年第12期1664-1669,共6页Basic and Clinical Medicine

基　　金：江苏省高校自然科学研究面上项目(14KJB310016);江苏省自然科学研究项目(By 2013042-06)

摘　　要：目的研究原发性肝癌(HCC)Hep G2细胞中EMS1/cortactin的表达与定位,探讨其功能特性。方法 RTq PCR、免疫荧光法检测EMS1基因和cortactin(EMS1编码蛋白)表达,共聚焦显微镜观察cortactin定位,并以人正常肝细胞L02为对照。将靶向EMS1的质粒EMS1-shRNA转染Hep G2,并以CCK8法、流式细胞周期检测、划痕和Transwell小室实验检测细胞增殖、周期、迁移和侵袭能力。结果 Hep G2中EMS1 mRNA水平为LO2的10.5倍;cortactin在Hep G2中高表达,并聚集于细胞膜下皮质区,L02中cortactin弥散分布在胞质中。与未处理组相比,转染EMS1-shRNA组Hep G2中EMS1 mRNA水平下降68.00%,cortactin水平下降52.80%,细胞增殖能力下降9.50%,S期比例下降37.89%,迁移能力下降45.40%,侵袭能力下降64.91%(均P<0.05)。结论 EMS1/cortactin主要与Hep G2细胞迁移和侵袭相关。Objective To explore expression and location of EMS1/cortactin in HCC cell line Hep G2. Methods RT-q QPCR was used to detect expression of EMS1. Location of cortactin（ protein encoded by EMS1 gene） was detected with immunofluorescence assay. L02（ normal liver cell） was used as control. Then a short-hairpin-RNA-expression plasmid against EMS1（ p S-EMS1） was constructed and transfected into Hep G2. The downregulation of EMS1 and cortactin were tested by RT-q PCR and Western blot analysis. The proliferation,division migration and invasion of Hep G2 were tested with CCK8 assay,flow cytometry assay,wound healing and Transwell invasion assays respectively. Results As compared with L02,the level of EMS1 gene in Hep G2 was 10. 5（ relative fold units）. Confocal microscopy showed that cortactin in Hep G2 accumulated to a high extent in the cell-substratum contact sites while it diffusely distributed in the cytoplasm in LO2. As compared with untreated group,RNAi-mediated suppression of EMS1/cortactin in Hep G2 cells was 68. 00% in mRNA level,and 52. 80% in protein level. InCCK8 assay,the inhibitiion rate was 9. 50% while in flow cytometry assay,reduction in the S phase was 37. 89%.Inhibition rates of migration and invasion were 45. 40% and 64. 91%（ P〈 0. 05） respectively. Conclusions EMS1/cortactin plays an important role in cellular invasive potential of HCC cell line Hep G2.

关 键 词：EMS1/cortactin 定位 迁移 侵袭 RNA干扰 

分 类 号：R735.7[医药卫生—肿瘤]