| Objective: Establishing human liver cancer cell SMMC-7721 model,to study and analyze the effect of HIF-1? or/and AKT protein expression,proliferation and apoptosis of liver cancer cells on knockdown of AKT or/and HIF1,and to explore the possible mechanism of AKT in HIF-1? signaling pathway.Methods: SMMC-7721 human hepatocellular carcinoma cell lines were cultured and randomly divided into five groups: blank control(BC),negative control(siRNA-NC),HIF-1 knockdown(HIF1-siRNA),AKT knockdown(AKT-siRNA)and HIF-1 and AKT knockdown(HIF1-AKT-siRNA)groups.Lentivirus interference technique was used to carry out transfection experiments on cells,and then the cell models with low expression of AKT or/and HIF-1 were constructed.The cells in BC group were not infected with lentiviruses,the cells in siRNA-NC group were infected with negative control lentiviruses,the cells in HIF1-siRNA group were infected with HIF-1 siRNA recombinant lentiviruses,the cells in AKT-siRNA group were infected with AKT siRNA recombinant lentiviruses,and the cells in HIF1-AKT-siRNA group were infected with HIF-1 siRNA and AKT siRNA recombinant lentiviruses.After stable transfection,the transfection efficiency was observed by inverted fluorescence microscopy;the inhibition rate of cell proliferation was detected by CCK-8 technology;the apoptotic level was detected by flow cytometry;and the expression of HIF-1alpha and AKT protein was determined by Western blot.Finally,according to the type and distribution of the data obtained,appropriate statistical methods were selected for processing and analysis.Results: The transfection efficiency of the experimental group was above 90%.Comparison of cell proliferation inhibition rate: siRNA-NC group(2.8±1.0)was a bit higher than BC group(0),but there was no significant difference between the two groups(P>0.05);HIF1-siRNA group(51.0±1.3),AKT-siRNA group(43.3±1.4)and HIF1-AKT-siRNA group(57.6±1.3)were significantly higher than siRNA-NC group,the difference was statistically significant(P<0.05);HIF1-siRNA group was higher than AKT-siRNA group,the difference was statistically significance(P<0.05);HIF1-AKT-siRNA group was higher than HIF1-siRNA group and AKT-siRNA group,the difference was statistically significant(P<0.05).Comparison of apoptosis levels: siRNA-NC group(11.51±0.25)was slightly higher than BC group(10.79±0.26),but there was no significant difference between the two groups(P>0.05);HIF1-siRNA group(54.83±.027),AKT-The siRNA group(21.81±0.24)and the HIF1-AKT-siRNA group(62.83±0.26)were significantly higher than the siRNA-NC group(P<0.05).The HIF1-siRNA group was higher than the AKT-siRNA group,the difference was significant(P<0.05);HIF1-AKT-siRNA group was higher than HIF1-siRNA group and AKT-siRNA group,the difference was statistically significant(P<0.05).HIF-1? protein expression levels were compared: siRNA-NC group(1.14±0.028)was slightly higher than BC group(1.08±0.048),the difference was not statistically significant(P>0.05);HIF1-siRNA group(0.42±0.021),AKT-siRNA group(0.92±0.019)and HIF1-AKT-siRNA group(0.27±0.018)were significantly lower than the siRNA-NC group(P<0.05);the HIF1-siRNA group was lower than the AKT-siRNA group,the difference was statistically significant(P<0.05);the HIF1-AKT-siRNA group was lower than the HIF1-siRNA group and the AKT-siRNA group,the difference was statistically significant(P<0.05).AKT protein expression levels were compared: siRNA-NC group(1.20±0.021)was slightly lower than BC group(1.23±0.020),but the difference did not achieve statistical significance.(P>0.05);HIF1-siRNA group(0.84±0.018),AKT-siRNA group(0.65±0.018)and HIF1-AKT-siRNA group(0.52±0.025)were significantly lower than the siRNA-NC group,the difference was statistically significant(P<0.05);the HIF1-siRNA group was higher than the AKT-siRNA group,the difference was statistical significance(P<0.05);HIF1-AKT-siRNA group was lower than HIF1-siRNA group and AKT-siRNA group,the difference was statistically significant(P<0.05).Conclusion: Knockdown of both HIF-1 and AKT expression could inhibit the proliferation and promote apoptosis of human liver cancer cell line smmc-7721.HIF-1 may be the primary growth factor of HCC cells,and HIF-1? expression level is enhanced under hypoxia.There is a bidirectional network regulation relationship between AKT and HIF-1.Knockdown of AKT expression can reduce HIF-1? translation level,inhibit cell proliferation and promote apoptosis.Conversely,knockdown of HIF-1 can also down-regulate AKT expression,inhibiting cell proliferation and promoting apoptosis.At the same time,knockdown of AKT and HIF-1 can play a synergistic role in inhibiting the proliferation of HCC cells and promoting apoptosis. |
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