Fak Expression In Liver Cancer Cell Line Smmc-7721 Experimental Study On The Effect Of Cellular Behavior

Hepotacellular Carcinoma (HCC) is one of the most common malignant tumors, the incidence about this cancer is rising steadily in most of countries. The treatment for HCC was still not improved with the development of diagnostic technique. Metastasis and recurrence is the main obstacle to the survival of the patients with HCC. To study the relationship of migation, proliferation and apoptosis of hepatoma carcinoma cell and progression of HCC is significance for the molecular target and preventing the recurrence of HCC and improving the therapeutic effect after surgical resection. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in signal transduction pathways that are initiated at sites of intrgrin-mediated cell adhesions and by growth factor receptors. Existed studies revealed that the over-expression of FAK was closely correlated with the metastasis of malignancy tumor. However, the molecular mechanism of HCC development and progression is unclear, and the research of FAK-siRNA in HCC is few until now. So we construct a FAK-siRNA expression vector and tansfect them into hepatoma carcinoma cells by opitimazed phosphate calcium transfection. The effect of FAK expression level on the migration, proliferation and apoptosis were studied, which can provide a new target and experiment data for gene therapy on HCC.Methods:1. We design the FAK specific siRNA expression vector based on nucleotide sequence of FAK supplied by GeneBank database and bioinformatics technique and construct the recombinant plasmids pU6H1-GFP-siFAK in vitro.2. Recombinant plasmids were transfected into SMMC-7721 with opitimazed phosphate calcium transfection.3. The groups:NC, pU6H1-GFP-siRNASCR, pU6H1-GFP-siFAK1, pU6H1-GFP-siFAK2, pU6H1-GFP-siFAK3, pU6H1-GFP-siFAK4. 4. FAK mRNA level was checked by semi-quantitative RT-PCR and protein expression was detected using western blotting and immunocytochemistry.5. The inhibitory effects on motility and proliferation were determined by wound healing assay and MTT, and cell apoptosis morphology was detected by Hoechast33258 staining.Results:1. The higher transfection efficiency was achieved with the optimized conditions 60h post transfection.2. The results of semi-quantitative RT-PCR and western blotting indicated, compared to NC group, FAK mRNA and protein level were significantly down-regulated (p<0.05). The inhibition rate of mRNA level of group F1 to F4 is 25.4%,62.4%,32.7% and 46.2% 48 hours post transfection. The expression of FAK protein decreased 31%,32.3%,38.5% and 35.8% 48h post tranfsfection in F1-F4 groups compared with NC group. The changes of FAK protein in immunocytochemistry also enhanced these conclusion.3. Wound healing assay, MTT and Hochast33258 staining:SMMC-7721 motility and proliferation ability were suppressed after pU6H1-GFP-siFAK transfection (p<0.05), and SMMC-7721 in F3 group showed typical apoptosis morphological features.Conclusions:FAK expression vector was successfully constructed. After SMMC-7721 cells transfection, it could effectively inhibit the expression of FAK. Decrease of FAK expression could significantly inhibit the ability of migration and proliferation in vitro, and promote apoptosis of SMMC-7721 cells.