OBJECTIVETo construct glypican-3 (GPC3)-green fluorescent protein eukaryotic expression vector pEGFP-c3-GPC3, and analyze the effect of GPC3 on the proliferation of human hepatoma cell line MHCC-97L.

METHODSThe eukaryotic expression vector pEGFP-c3-GPC3 was constructed with recombinant DNA technique and transfected into MHCC-97L cells via Lipofectamine 2000. The cells stably expressing GPC3 were screened by flow cytometry and G418. The mRNA expression of GPC3 was detected by RT-QPCR method, and the protein expression by Western blotting and fluorescence microscope. The effect of GPC3 gene on the growth of the cells was examined by MTT assay.

RESULTSRestriction endonuclease analysis and DNA sequencing verified correct construction of the recombinant plasmid. The green fluorescence was detected in the transfected MHCC-97L cells under fluorescence microscope. RT-QPCR and Western blotting both confirmed successful expression of GPC3 in MHCC-97L cells. The growth curve showed a significant acceleration of the proliferation of the transfected MHCC97-Lsol;GPC3 cells as compared with MHCC97-L and MHCC97-L/C3 cells (P<0.001).

CONCLUSIONWe have successfully constructed the eukaryotic expression vector pEGFR-c3-GPC3, which allows stable GPC3 expression in MHCC97-L/GPC3 cells. The upregulation of GPC3 expression can stimulate the growth of hepatoma cell line MHCC97-L in vitro.

Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Genetic Vectors ; Glypicans ; pharmacology ; Green Fluorescent Proteins ; genetics ; Humans ; Liver Neoplasms ; pathology ; Plasmids ; Transfection