Screening Of Human Hepatocellular Carcinoma Cell Lines With High Expression Of Gcf2Gene And The Research About Sirna Mediated Gcf2Gene Silencing

Objective:To screen of human hepatocellular carcinoma cell lines with the high expression of GCF2gene. Meanwhile, to screen of siRNA with the high interference efficiency so as to to investegate its effect on the apoptosis of the hepatocellular carcinoma cell line.Methods:(1) The expression of GCF2gene in four human hepatocellular carcinoma cell lines (BEL-7404, HepG2, SMMC-7721, QGY-7703) and one human normal liver cell line (HL-7702) were assayed by RT-PCR, western blot and immunohistochemistry technique.(2) Designing5siRNA segments targeting the GCF2gene. These GCF2siRNA segments were transfected into BEL-7404by means of Lipofectamine2000respectively. There were eight groups in this study including siRNA GCF2-1group, siRNA GCF2-2group, siRNA GCF2-664group, siRNA GCF2-863group, siRNA GCF2-919group, negative control group, Lipofectamine group and blank control group. Semi-Quantitative RT-PCR and western blot were used to dimension the GCF2 expression at mRNA and protein levels, respectively.(3) The cell apoptosis were evaluated by flow cytometry.Result:(1) Semi-Quantitative RT-PCR results showed that the expression of GCF2mRNA in BEL-7404cell line was the lowest (P<0.001). Oppositely, the expression of GCF2protein in BEL-7404cell line was the highest by western blot (P=0.015) among. IHC results showed that GCF2proteins were expressed both in cell nuclear and in the cytoplasmic. The expression of GCF2protein in BEL-7404, HepG2, SMMC-7721, QGY-7703was higher than those in HL-7702, among them with BEL-7404expression of the highest amount.(2) Compared with negative control group, Lipofectamine group and blank control group, the expression of GCF2mRNA and protein were decreased significantly in siRNA GCF2-1group, siRNA GCF2-664group, siRNA GCF2-863group and siRNA GCF2-919group(P<0.05). It’s important to realize, the expression of GCF2mRNA and protein in siRNA GCF2-1group was the lowest among above.(3) siRNA induced apoptosis of BEL-7404cells with the apoptosis rate of15.633±2.650, while the rate in the control group was7.767±0.757by Annexin V-EGFP/PI double staining flow cytometry. This suggested that GCF2played a role in apoptosis of BEL-7404cell line.Conclusion:The expression of GCF2mRNA in BEL-7404cell line was the lowest; the expression of GCF2protein in BEL-7404cell line was the highest. GCF2proteins were expressed both in cell nuclear and in the cytoplasmic by IHC. The interference efficiency of siRNA GCF2-1was63.3%, the siRNA induced apoptosis of BEL-7404cells. Consequently, GCF2gene may regulate the human hepatocellular carcinoma cell lines apoptosis.