蛋白激酶B通过调控低氧诱导因子-1表达干预人肝癌细胞系SMMC-7721增殖

AKT intervention on the proliferation of human hepatoma cell line SMMC-7721 through regulating HIF-1 expression

目的研究与分析敲低蛋白激酶B(PKB/AKT)对低氧诱导因子-1(HIF-1)表达及肝癌细胞增殖的影响。方法随机将人肝癌细胞系SMMC-7721分设5组:空白对照(BC)组、阴性对照(siRNA-NC)组、HIF-1 siRNA转染(HIF1-siRNA)组、AKT siRNA转染(AKT-siRNA)组和HIF-1 AKT siRNA转染(HIF1-AKT-siRNA)组。用倒置荧光显微镜观察转染效率;CCK-8法检测细胞增殖抑制率;Western blot检测HIF-1蛋白的表达。结果转染效率均达到90%以上。与siRNA-NC组相比较,HIF1-siRNA组、AKT-siRNA组和HIF1-AKT-siRNA组的细胞增殖抑制率均升高,HIF-1蛋白表达水平均下降(P<0.05);与HIF1-AKT-siRNA组比较,HIF1-siRNA组和AKT-siRNA组的细胞增殖抑制率均下降,HIF-1蛋白表达水平均升高(P<0.05);与HIF1-siRNA组比较, AKT-siRNA组的细胞增殖抑制率下降,HIF-1蛋白表达水平升高(P<0.05)。各组HIF-1蛋白表达水平与细胞增殖抑制率存在负相关关系(r=-0.874,P<0.05)。结论敲低HIF-1、AKT表达均可抑制人肝癌细胞系SMMC-7721增殖;HIF-1可能是肝癌细胞增殖的主要决定因子, HIF-1表达水平下调可抑制肝癌细胞增殖;敲低AKT表达可降低HIF-1表达水平,并协同HIF-1低表达的抑肝癌细胞增殖作用。

Objective The effect of knockdown of AKT expression on the proliferation of hepatocellular carcinoma (HCC) and its regulation was analyzed. Methods SMMC-7721 cells were transfected with recombinant lentivirus contained a HIF-1 siRNA and/or AKT siRNA. Then, the transfection efficiency was observed under inverted fluorescence microscope, the cell growth inhibition rate was detected by cell counting kit-8 (CCK-8), the expression level of HIF-1 protein was detected by Western blot, and the protein expression of HIF-1 was analyzed statistically. Results SMMC-7721 cells was transfected with siRNA-lentivirus with ≥90% efficiency. The inhibition rate of HIF1-siRNA group, AKT-siRNA group, and HIF1-AKT-siRNA group decreased significantly as compared to the siRNA-NC group ( P <0.05), and the expression of HIF-1 proteins was significantly higher. On the other hand, the inhibition rate of HIF1-AKT-siRNA group was significantly higher as compared to the HIF1-siRNA group and AKT-siRNA group, and the expression of HIF-1 proteins decreased significantly ( P <0.05). While the inhibition rate in the HIF1-siRNA group was significantly higher as compared to the AKT-siRNA group, and the expression of HIF-1 proteins decreased significantly. There was a negative correlation between the expression of HIF-1 protein and the inhibition rate of cell proliferation ( r =-0.874, P <0.05). Conclusions The downregulation of HIF-1 or AKT expression inhibits the cell proliferation in SMMC-7721 cells. HIF-1 plays a vital role in the cell proliferation in human hepatoma cell, down-regulation of HIF-1 protein expression can inhibit human hepatoma cell proliferation. The knockout of AKT resultes in down-regulation of the protein expression of HIF-1 and intervenes on the proliferation of human hepatocellular carcinoma synergizing with down-regulation of HIF-1.