HIF-1α基因沉默对人肝癌SMMC-7721细胞VEGF和MMP-2基因表达的影响

The influence of silencing HIF-1α gene on expressions of VEGF gene and MMP-2 gene in SMMC-7721 cell line

目的探讨缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)基因沉默对人肝癌SMMC-7721细胞中血管内皮生长因子(vascular endothelial growth factor,VEGF)和基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)基因表达的影响。方法构建HIF-1α基因的RNA干扰(RNAi)表达载体(HIF-1α-shRNA-pGenesil-1)和阴性对照载体(shRNA-HK-pGenesil-1),并将其与质粒结合。将人肝癌SMMC-7721细胞分为沉默组、阴性对照组和空白对照组,分别转染HIF-1α-shRNA-pGenesil-1重组载体、shRNA-HK-pGenesil-1重组载体和空载体pGenesil-1。转染细胞经浓度为500μg/mL的G418溶液筛选后获得带有转染重组载体的细胞克隆,分别于常氧、低氧6 h、低氧12 h及低氧24 h条件下培养,再采用实时定量逆转录聚合链式反应(RT-PCR)检测细胞中HIF-1α mRNA、VEGF mRNA及MMP-2 mRNA的表达。结果常氧条件下,沉默组、阴性对照组和空白对照组细胞中均无HIF-1α mRNA的表达,且3组细胞中VEGF mRNA及MMP-2 mRNA的表达水平比较差异均无统计学意义(P>0.05)。低氧培养6、12及24 h时,沉默组细胞中HIF-1α mRNA、VEGF mRNA及MMP-2 mRNA的表达水平均低于同时点空白对照组和阴性对照组(P<0.05),但同时点空白对照组和阴性对照组比较差异均无统计学意义(P>0.05)。与常氧条件对应组别比较,沉默组、阴性对照组和空白对照组各低氧条件组细胞中HIF-1α mRNA、VEGF mRNA及MMP-2 mRNA的表达水平均较高(P<0.05);低氧条件下,3组细胞中HIF-1α mRNA、VEGF mRNA及MMP-2 mRNA的表达水平均呈下降趋势,同组内3个时点间两两比较差异均有统计学意义(P<0.05)。结论利用RNAi技术有效沉默人肝癌SMMC-7721细胞中HIF-1α基因的表达后,VEGF mRNA和MMP-2 mRNA的表达水平均降低,提示沉默HIF-1α基因可抑制原发性肝癌的浸润和转移。

Objective To explore the influence on the expressions of vascular endothelial growth factor （VEGF） gene and matrix metalloproteinase-2 （MMP-2） gene in hepatocellular carcinoma of SMMC-7721 cells with RNA interference （RNAi） silencing the expression ofhypoxia inducible factor-1α （HIF-1α） gene. Methods Firstly, constructed short hairpin RNA （shRNA） targeting for HIF-1α gene, and then transfected it to SMMC-7721 cells after combining with plasmid. The SMMC-7721 cells were divided into three groups, silencing group, negative control group, and blank control group, which were transfected with HIF-1α-shRNA-pGenesil- 1 recombinant vector, shRNA-HK-pGenesil- 1 recombinant vector, and pGenesil-1 vector respectively. Transfection cells were screened by the concentration of 500 μg/mL G418, and then positive and negative cell clones with transfection recombination carrier were obtained. Detected the expressions of HIF-1α mRNA, VEGF mRNA, and MMP-2 mRNA in the 3 groups with real time PCR （RT-PCR） technology, under the condition of hypoxic training 6 h, 12 h, and 24 h, as well as conventional oxygen training. Results There was no expression of HIF-1α mRNA at conventional oxygen condition in the 3 groups, and there was no significant difference in expressions of VEGF mRNA and MMP-2 mRNA among the 3 groups （P〉0.05） at the condition of conventional oxygen training. The expressions of HIF-1α mRNA, VEGF mRNA, and MMP-2 mRNA in the silencing group, compared with the the negative control group and the blank control group, were obviously decreased （P〈0.05） under the condition of hypoxic training （6, 12, and 24 h）, while there was no significant difference between the negative control group and the blank control group at each time point （P〉0.05）, but the expressions of HIF-la mRNA, VEGF mRNA, and MMP-2 mRNA in the 3 groups under every condition of hypoxic training were all higher than those of conventional oxygen condition （P〈0.05）. Under the condition of hypoxic training, the expressions of HIF-1α mRNA, VEGF mRNA, and MMP-2 mRNA in the 3 groups decreased over time, and there was significant difference between any 2 time points in each group （P〈0.05）. Conclusion RNAi technique can effectively silence the expression ofHIF-1α mRNA of SMMC-7721 cells, and then silence the expressions of VEGF and MMP-2 mRNA, to inhibit the invasion and metastasis of hepatocellular carcinoma.