| Objective We intervened Wipl gene in human hepatoma cell line Huh-7 by transfecting target plasmid or siRNA in order to increase or decrease the expression of Wip1 gene. After verified that Wip1 gene was intervened, we further observed the expression of wipl protein, the proliferative capacity of Huh-7, the migration capacity of Huh-7, the invasive capacity of Huh-7. We tried to preliminary study the reason and mechanism of these changes so that this experiment could offer theoretical support for our further animal experiment and supply new methods and ideas for the treatment and intervention of hepatic carcinoma in clinical.Methods We transfected target plasmid or siRNA into human hepatoma cell line Huh-7 by NanoFection Transfection Reagent(ExCell). The target plasmid could increase the expression of Wipl gene, while the target siRNA could decrease the expression of Wip1 gene by silencing the mRNA related to Wip1 gene. We did PCR experiment to make sure whether the expression of Wip1 gene related mRNA had changed 24h after transfection was done. If it did change, we did western blot to test whether the expression of wipl protein had changed 48h after transfection. CCK-8 experiment was done to test the changes of proliferative capacity of Huh-7 24h after transfection and we observed the results at 24h,48h,72h. Cell scratch experiment was done 24h after transfection in order to text the changes of migration capacity of Huh-7 and we observed the results at 24h,48h,72h. Transwell experiment was done to test the changes of invasive capacity of Huh-7 24h after transfection and we observed the results at 24h.Results The expression of Wipl gene in Huh-7 had increased after the plasmid transfection was done 24h later. The expression of wipl protein also increased 48h later after transfection was done. In the CCK-8 experiment, we found that the OD values of plasmid transfection group were larger than the untransfected group, no matter in different time groups or in the different cell number groups. The proliferative rates of plasmid transfection group were between 107.42% and 176.36%, while most of the proliferative rates were near 130%. In the cell scratch experiment, the untransfected group’s migration distance was nearer than the plasmid transfection group in all different time groups. The experiment results were as follows,24h group: 34.78±4.77μm VS 61.60±2.02μm(t=8.97, P<0.05),48h group:33.98±3.48μm VS 64.75±4.67μm(t=9.19, P<0.05),72h group:35.49±0.90μm VS 67.89±4.23μm(t=13.01,P<0.05). In the transwell experiment, we found that more plasmid transfectd Huh-7 cell invaded into the lower chamber. The three times of experiment results were as follows, the first time:90.00±3.00 VS 178.67±5.77(t=23.65,P<0.05), the second time:74.33±26.95 VS 209.00±32.91(t=5.48,P<0.05), the third time:79.33±20.74 VS 208.67±73.24(t=13.01,P<0.05).The expression of Wipl gene in Huh-7 had decreased after the siRNA transfection was done 24h later. The expression of wip1 protein also decreased 48h later after transfection was done. In the CCK-8 experiment, we found that the OD values of siRNA transfection group were smaller than the NC group, no matter in different time groups or in the different cell number groups. The inhibition rates of siRNA transfection group were between 12.37% and 40.81%, while most of the inhibition rates were near 30%. In the cell scratch experiment, the NC group’s migration distance was farer than the plasmid transfection group in all different time groups. The experiment results were as follows,24h group:31.24±4.42μm VS 16.04±6.21μm(t=18.53, P<0.05),48h group:31.33±2.13μm VS 18.20±5.67μm(t=16.09,P<0.05),72h group:32.45±3.08μm VS 18.99±5.32μm(t=16.50,P<0.05). In the transwell experiment, we found that less siRNA transfected Huh-7 cell invaded into the lower chamber. The three times of experiment results were as follows, the first time:74.00±12.17 VS 33.00±10.44(t=14.91,P<0.05), the second time:77.00±5.00 VS 32.67±2.08(t=22.05,P<0.05), the third time:81.33±17.16 VS 29.30±2.51 (t=20.32,P<0.05).Conclusion After the target plasmid transfection, the expression of Wip1 gene and wip1 protein in Huh-7 both increased and the proliferative capacity, the migration capacity, the invasive capacity were all increased as well. After the target siRNA transfection, the expression of Wipl gene and wipl protein in Huh-7 both decreased and the proliferative capacity, the migration capacity, the invasive capacity were all decreased too. |
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