Influence Of GPC3 Interfered By RNA Interference On Biological Behavior Of Huh-7 Cells

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors which seriously threat the body health of human being. China is among the high incidence area of HCC, with an incidence of 20.37 per 100 thousand which is the third highest incidence in common tumors. According to the statistical data collected in 2002, HCC's incidence ranked the sixth in common cancers while its mortality ranked the third; the annual incidence cases of HCC was 626 thousand which showed a 5.7% increase, while the number of death was 598 thousand, among which 82% of the cases appeared in developing country, especially China enjoyed 55%. Though many methods including surgical resection, liver transplantation, vascular intervention, ablation technology, radiotherapy, chemotherapy, can be performed in HCC in clinic practice,more than one half of HCC patients had the micrometastasis failing to dectect before radical surgery,which may be the direct reason of metastasis or recurrence of HCC after surgery. Metastasis and recurrence have become the crux of further improvement on survival rate of HCC patients. Therefore, to seek and predicate HCC's metastasis and recurrence tendency, judge prognosis of HCC patients is the key and focus of HCC's research, as well as its emphasis and difficulty. Glypican-3(Glypican-3, also called MXR7, OCI-5, and GTXR2-2) is a member of HSPG (heparan sulfate proteoglycan) family. In the way of combining with heparin binding proteins such as cell adhesion molecules, matrix components, growth factors, enzyme and enzyme inhibitors, GPC3 participates in the control of cell proliferation, differentiation, adhesion, migration and so on, even possibly involved in the inhibition or control of most tissues and organs' growth in mesoderm. The mutation of GPC3 leads to imbalance of cell proliferation and apoptosis, which may be the key reason of tumor development. So far research shows that GPC3 participates in the control of signal transduction pathway which closely related to tumor development such as Wnt,Hedgehog (Hh),FGF,IGF,BMP,SMAD,TGF-βand so on. Researches also show that GPC3 is highly expressed in HCC, colorectal carcinoma,Wilm's tumor, neuroblastoma and hepatoblastoma, while loses its expression in lung adenocarcinoma, Ovarian cancer, breast cancer, mesothelioma, which suggests that GPC3 is a worth-expecting specific marker of multiple tumors.Double-stranded RNA (dsRNA) can induce gene-silencing processes in eukaryotes through the degradation of homologous mRNAs, a process known as RNA interference. Small interference RNA(siRNA) or shot hairpin RNA(shRNA) both are the medium of RNAi.There are several characters of RNAi,including specificity of sequence which can discriminate mutation gene sequence with normal gene sequence,high efficiency and amplification effect.shRNA is more stable than siRNA and also can extent the time of target gene silencing.This research intent to test GPC3's influence on biological behavior of huh-7 cells by transient transfecting the GPC3-targeted siRNA into huh-7 cells and observing the changes of huh-7 cells on cell proliferation,cell apoptosis and cell migration.The object of the study is to provide the evidence to the feasibility of hepatocellular carcinoma gene therapy. Design GPC3-targeted siRNA,transient transfect huh-7 cells,and detect the intracellular expression of GPC3 gene.1. Design of siRNA Apply siRNA design software for screening out of nine pairs of siRNA,and then remove the siRNA which are homologous with the other encoding genes,the final select two pairs of GPC3-targeted siRNA.2. Transient transfection Transfect GPC3-siRNA into huh-7 cells by lipofection technique,and detect after 48h.3. Detecting Expression of GPC3 mRNA was detected by real-time PCR,expression of GPC3 protein was identified by Western blot,cell apoptosis rate was evaluated by flow cytometry, cell proliferation was detected by MTT assay,and cell migration was detected by wound healing assay.4. Statistical methods The difference of GPC3 protein and mRNA expression rate was analyzed by Chi-square test and Wilcoxon rank sum test respectively.The difference among groups on cell proliferation,cell apoptosis and cell migration was analyzed by Factorial design analysis of variance.1. Expression of GPC3 mRNA The result showed that the expression of GPC3 mRNA of siRNA interfered huh-7 cells of two groups was further lower than control huh-7 cells group.2. Expression of GPC3 protein The result displayed that protein level of siRNA transfected huh-7 cells of two groups was lower than control huh-7 cells group.3. Apoptosis The result of 48hrs and 72hrs after transfection demonstrated that apoptosis rate of two experimental groups was higher than control group.However,there are smaller difference between two experimental groups and control group at the time point of 96hrs. 4. Cell proliferation The result of 48hrs and 72hrs after transfection also show that OD490nm detected by Spectrophotometer of two experimental groups is lower than of control group.Small difference can be seen in 96hrs among groups.5. Cell migration The result of wound healing assay displayed that the number of migratiing cells of experimental groups were significantly lower than the control group.1. Three-step method of design siRNA is very feasible and effective.2. Application of RNAi technology,GPC3-targeted siRNA can induce GPC3 gene silencing to achieve the purpose of closure of GPC3 gene.The results showed that the biological behavior of huh-7 cells interfered by GPC3-targeted siRNA changed significantly,including decline of cell proliferation and cell migration and increase of cell apoptosis.lt suggested that GPC3 gene may play a key role on regulation of biological behavior of hepatoma cells.3. Transient transfection of GPC3-targeted siRNA can efficiently knock out the GPC3 gene expression of huh-7 cells,but only last for a short time.