EBLN2基因对肝癌细胞增殖的抑制作用及其机制

摘要

Objective To study the inhibitory effects of endogenous Bornavirus-like nucleoprotein 2 (EBLN2)gene on proliferation of hepatocellular carcinoma (HCC)cell lines and its mechimasm.Methods The real-time fluorescent quantitative RT-PCR and Western blotting was used to detect the expression of EBLN2 in HCC cell lines (HepG2,SK-hep-1,Huh7,Hep3B,MHCC-97H,PLC /PRF /5,Focus,SMMC7721,YY-8103 and LO2)and human HCC tissue samples. The overexpression of EBLN2 was carried out in Focus and YY-8103 cells with low expression of EBLN2.The recombinant plasmid pCMV-EBLN2 was transfected into Focus and YY-8103 cells.At 24 h,the effects of overexpression of EBLN2 gene on the proliferation of HCC cell lines,plate clone formation ability,soft agar clone formation ability,and cell cycle.Re-sults The quantitative and semi-quantitative PCR results showed that EBLN2 mRNA expression was down-regulated in 59% HCC samples.Further,compared with the normal hepatic tissues,EBLN2 expression was down-regulated in cell lines such as HepG2,SK-hep-1,Huh7,Hep3B,MHCC-97H,PLC /PRF /5,Focus,SMMC7721,YY-8103 and LO2 with the percentage of 48%,73%,72%,79%,84%,72%,87%,74%,82% and 80%,respectively (all P <0.05).After the Pcmv-EBLN2 plasmid was transferred into cell lines YY-8103 and Focus for 24 h,the proliferation rates of YY-8103 and Focus cell lines were 72.2% and 85%.The plate clone formation rates of YY-8103 and Focus cells were 52.6% and 25.6%,and the soft agar clone formation rates of YY-8103 and Focus cells were 40.9% and 37.5%,and significant difference was found,all P <0.05.The cell cycle transition from G1 phase to S phase was inhibited in the YY-8103 and Focus cells.Conclusions EBLN2 is down-regulated in HCC tissues and inhibits the proliferation of HCC cells by regula-ting the cell cycle.%目的：探讨人类内源性非逆转录博纳病毒样核蛋白序列2（EBLN2）基因对肝癌细胞增殖的抑制作用及其机制。方法采用实时定量 RT-PCR 技术和蛋白印迹技术检测 EBLN2基因在肝癌细胞系（HepG2、SK-hep-1、Huh7、Hep3B、MHCC-97H、PLC ／PRF ／5、Focus、SMMC7721、YY-8103、LO2）和人肝癌组织样本中的表达；选取 EBLN2表达水平较低的肝癌细胞株 Focus 和 YY-8103进行外转质粒过表达，Pcmv-EBLN2重组质粒转入肝癌细胞株 Focus和 YY-8103，24 h 观察 EBLN2基因过表达对肝癌细胞株细胞增殖、平板克隆形成能力及软琼脂克隆形成能力、细胞周期的影响。结果定量和半定量 PCR 实验结果表明 EBLN2基因在59％的肝癌样本中表达下调，且在10株肝癌细胞系（HepG2、SK-hep-1、Huh7、Hep3B、MHCC-97H、PLC ／PRF ／5、Focus、SMMC7721、YY-8103、LO2）中表达水平与正常肝组织相比明显下调，依次下调48％、73％、72％、79％、84％、72％、87％、74％、82％、80％，P 均＜0．05；Pcmv-EBLN2质粒转染肝癌细胞株 YY-8103和 Focus 24 h 后，肝癌细胞株 YY-8103和 Focus 的细胞增殖率分别为72．2％、85．0％，平板克隆形成率分别为52．6％、25．6％，软琼脂克隆形成率分别为40．9％、37．5％，与空白对照相比，P 均＜0．05；肝癌细胞株 YY-8103和 Focus 从 G1期进入 S 期受到抑制。结论 EBLN2基因在肝癌组织中表达下调，其可能通过调节细胞周期抑制肝癌细胞增殖。