Construction Of GPC3 Green Fluorescent Protein Eukaryotic Expression Vector And Effects Of GPC3 On SK-Hep-1 Hepatocellular Carcinoma Cells

GPC3 have an intimate relationship with kinds of tumors, including liver cancer, malignant melanoma, ovarian cancer and breast cancer. It plays different roles in different tumors, even an opposite effect. GPC3 may plays very important biological functions, particularly in liver cancer. The target gene was transfected into specific cells through gene transfection technology to observe the functions of GPC3 in SK-Hep-1. This is the broadest applications, and the most sophisticated gene technology research methods. In this study, GPC3 eukaryotic expression vector with enhanced green fluorescent protein marker was constructed. through using gene transfection and tumor invasion and migration technology, The impacts of GPC3 on biological behavior of liver cancer cell was observed from protein and gene level in vitro experiments. Effects of growth factors on GPC3-SK-Hep-1, including FGF2 and IGF2, were also observed here toilluminate if GPC3 regulated growth factor signaling.一,Construction of GPC3 green fluorescent protein eukaryotic expression vectorAccording to the GenBank GPC3 open reading frame(1740bp), the primerswere designed and GPC3 gene fragments were cloned through PCR method. PCR products and pEGFP-N2 plasmid were cutted by XhoⅠand EcoRⅠenzyme.Then they were connected. After transformation, the clone selected was delivered to detect sequencing, GPC3 green fluorescent protein eukaryotic expression vector was successfully constructed.二,The expression of GPC3 in SK-Hep-1 liver cancer cells and the involvement of function of GPC3 in SK-Hep-1 After the GPC3 green fluorescent protein eukaryotic expression vector was successfully constructed, SK-Hep-1 cells were transfected with pEGFP-N2-GPC3 and control vector (pEGFP-N2) using Lipofectamine2000. Transfected SK-Hep-1 cells were selectively screened with G418 (600μg/ml). The cells were named GPC3-SK-Hep-1 and pEGFP-SK-Hep-1(control cells), as well as SK-Hep-1(blank cells) is erected. GPC3mRNA level was detected by RT-PCR. After GPC3 was transfected into SK-Hep-1 cells successfully. The morph feature of three groups cells were observed through microscope. The growth curve of three groups of cells GPC3-SK-Hep-1, pEGFP -SK-Hep-1, SK-Hep-1 were drawed through MTT colorimetric assay methods(P<0.001 compared with blank and control).We analyzed the effects of GPC3 on SK-Hep-1 cells in cell adhesion, migration and invasion in vitro. The results displayed that forced expression of GPC3 reduced adhesion capacity(P<0.01 compared with blank and control), but stimulated migration and invasion capacity. In adhesion experiment, The SK-Hep-1 cells expressing GPC3 displayed significantly reduced adhesion to matrigel. The adhesion rate of GPC3-SK-Hep-1 cells were (10.21±0.62)%, and in blank plasmid transfected cells were (15.13±0.83)%. The inhibition rate was 34.17%. In migration experiment, the trans-membrane cell numbers in GPC3 transfected SK-Hep-1 hepatoma carcinoma cells were (131.7±7.44)/HT, and in blank plasmid transfected cells were (71.6±4.76)/HT. In invasion experiment, the trans-membrane cell numbers in GPC3 transfected SK-Hep-1 hepatoma carcinoma cells were (220±12.8)/HT, and in blank plasmid transfected cells were (138±10.5)/HT. There was significant difference between the two groups(P<0.001). Effects of growth factors on GPC3-SK-Hep-1 were detected by MTT. FGF2-induced cell proliferation proved to be significantly decreased by GPC3(P<0.05), whereas the growth effects of IGF2, was not altered by GPC3(P>0.05), It suggested that GPC3 regulated growth factors signaling.