EGFL8基因沉默对肝癌细胞侵袭转移能力的影响

The impact of EGFL8 gene silencing on the invasion and metastasis of hepatocellular carcinoma cells

目的观察EGFL8基因沉默对Hep3B人肝癌细胞系侵袭迁移能力的影响。方法采用实时定量PCR方法检测EGFL8基因在Hep3B、SMMC-7721和HCCLM3这3种具有不同转移潜能的肝癌细胞系中的表达水平。采用慢病毒介导的小干扰RNA(Small interfering RNA;siRNA)方法构建了EGFL8基因沉默的Hep3B细胞系,实验分为shEGFL8组(实验组)和shCtrl组(对照组),以实时定量PCR法检测了EGFL8基因的沉默效率,并以划痕愈合和Transwell小室侵袭实验观察EGFL8基因沉默对Hep3B细胞系侵袭迁移能力的影响。结果EGFL8基因表达水平在低转移潜能的Hep3B肝癌细胞系中最高(0.002106±0.000782),在中等转移潜能的SMMC-7721细胞系中次之(0.0006028±0.00003128),在高转移潜能的HCCLM3细胞系中最低(0.0002613±0.000019)。shEGFL8组中的EGFL8表达水平明显低于shCtrl组(0.233±0.007比1.013±0.118,t=6.582,P=0.0028)。Transwell小室侵袭实验结果显示,shEGFL8组Hep3B细胞穿过聚碳酸酯膜的平均细胞数明显高于shCtrl组(105.3±6.983 vs.52.33±4.256,t=6.48,P=0.0029)。划痕愈合实验中,shEGFL8组Hep3B细胞的迁移率明显高于shCtrl组(24 h迁移率:10.67%±1.20%vs.5.67%±0.33%,t=4.009,P=0.016;(72 h迁移率:22.67%±1.20%vs.12.67%±1.20%,t=5.883,P=0.004)。结论EGFL8基因下调与肝癌细胞系的转移潜能相关,EGFL8基因沉默可显著增强Hep3B肝癌细胞系的侵袭迁移能力,提示EGFL8是一个肝癌转移抑制基因。

Objective To observe the effect of EGFL8 gene silencing on invasion and migration of human hepatocellular carcinoma cells Hep3 B.Methods Real-time quantitative PCR was used to detect the expression of EGFL8 gene in hepatocellular carcinoma cells Hep3 B、SMMC-7721 and HCCLM3,which had different metastasis potential.Lentiviral-mediated small interfering RNA(siRNA)inhibiting EGFL8 gene was transfected in hepatocellular carcinoma Hep3 B cells.The experiment was divided into shEGFL8 group(experimental group)and shCtrl group(control group).Real-time quantitative PCR was used to detect EGFL8 gene silencing effect.The impact of EGFL8 gene silencing on invasion and migration of Hep3 B cells was determined by Wound healing and Transwell chamber invasion assay.Results The expression of EGFL8 gene was the highest in hepatocellular carcinoma cells Hep3 B with weak metastatic potential(0.002106±0.000782),followed by hepatocellular carcinoma cells SMMC-7721 with medium metastatic potential(0.0006028±0.00003128),and the lowest in hepatocellular carcinoma cells HHCCLM3 with strong metastatic potential(0.0002613±0.000019).The expression of EGFL8 in the shEGFL8 group was significantly lower than that in the control group(0.233±0.0071.013±0.118,t=6.582,P=0.0028).Transwell chamber invasion assay showed that the average cell number of Hep3 B cells passing through the polycarbonate membrane in the shEGFL8 group was significantly higher than that in the control group(105.3±6.9852.33±4.256,t=6.48,P=0.0029).In the wound healing assay,the cell migration distance of the shEGFL8 group was significantly higher than that of the control group(24 h migration ratio:10.67±1.205.67±0.33,t=4.009,P=0.016;72 h migration ratio:22.67±1.20 vs.12.67±1.20,t=5.883,P=0.004).Conclusion Down-regulation of EGFL8 gene is associated with the metastatic potential of hepatocellular carcinoma cells.Silencing EGFL8 gene can significantly enhance the invasion and migration of hepatocellular carcinoma cells Hep3 B,suggesting that EGFL8 is a hepatocellular carcinoma metastasis suppressor gene.