过表达Glypican-3对高转移性肝癌细胞HCCLM3r生物学行为的影响

摘要

旨在构建sGPC3/GPC3基因重组真核表达载体,研究过表达sGPC3/GPC3对HCCLM3人高转移性肝癌细胞生物学行为的影响.采用PCR法获得编码sGPC3/GPC3的基因片段并克隆到真核表达载体pcDNA3.0-GFP中,经脂质体介导转染HCCLM3肝癌细胞,RT-PCR和Western blot检测GPC3的表达情况;CCK-8法、克隆平板形成实验、细胞划痕实验、流式细胞术分别检测sGPC3/GPC3对HCCLM3生物学行为的影响.酶切分析、测序鉴定结果表明sGPC3/GPC3基因重组真核表达载体构建成功;RT-PCR和Western blot结果显示sGPC3/GPC3在HCCLM3中成功过表达.与正常组和转染空载体相比,过表达sGPC3/GPC3后,HCCLM3的增殖能力和克隆形成能力均受到明显抑制(P<0.05),迁移率显著下降,细胞周期阻滞于G1期,细胞凋亡率增加.同时,过表达sGPC3对细胞的影响显著优于GPC3(P<0.05).sGPC3/GPC3表达上调抑制高转移性肝癌细胞HCCLM3生长和迁移,促进细胞凋亡以及周期再分布,在高转移性肝癌发生发展过程中起重要作用.%This work aims to construct the recombinant eukaryotic expression vector for sGPC3/GPC3(soluble glypican-3/glypican-3) gene,and to study the effects of overexpressing sGPC3/GPC3 on the biological behaviors of human highly metastatic hepatocarcinoma cells HCCLM3. The gene fragment encoding sGPC3/GPC3 was obtained by PCR and cloned into eukaryotic expression vector pcDNA3.0-GFP,then with which HCCLM3 was transfected by liposome-mediated method. The expression of GPC3 was detected by RT-PCR and Western blot. The effects of sGPC3/GPC3 on the biological behavior of high metastatic hepatocarcinoma cells were detected by CCK-8,clonal plate formation experiment,cell scratch test and flow cytometry. Results showed that the recombinant eukaryotic expression vector of sGPC3/GPC3 gene was successfully constructed by restriction enzyme digestion and sequencing. The results by RT-PCR and Western blot demonstrated that sGPC3/GPC3 was successfully overexpressed in HCCLM3. Compared with normal group and transfected control vector,the proliferation ability and clonogenic ability of HCCLM3 were significantly inhibited by the overexpression of sGPC3/GPC3(P < 0.05),the mobility significantly decreased and the cell cycle was arrested in G1 phase,thus,the rate of apoptosis increased. Moreover,the effect by overexpressing sGPC3 was significantly better than by GPC3(P < 0.05). Conclusively,the up-regulation of sGPC3/GPC3 inhibits the growth and migration of highly metastatic hepatocarcinoma cells HCCLM3,induces the cell apoptosis and affects the redistribution of cell cycle,which plays an important role in the development of highly metastatic liver cancer.