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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 investigate the effects of hypoxia inducible factor-1α (HIF-1α) gene silencing on the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) -2, MMP-2) gene expression. Methods RNAi expression vector (HIF-1α-shRNA-pGenesil-1) and negative control vector (shRNA-HK-pGenesil-1) of HIF-1α gene were constructed and combined with plasmid. The human hepatocellular carcinoma SMMC-7721 cells were divided into two groups: silence group, negative control group and blank control group, transfected with HIF-1α-shRNA-pGenesil-1 recombinant vector, shRNA-HK-pGenesil-1 recombinant vector and empty vector pGenesil-1 . The transfected cells were screened by G418 solution with a concentration of 500μg / mL to obtain the cell clones with the recombinant vector transfected, respectively, under normoxia, hypoxia for 6 h, hypoxia for 12 h and hypoxia for 24 h, Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of HIF-1α mRNA, VEGF mRNA and MMP-2 mRNA. Results No expression of HIF-1α mRNA was detected in the silenced group, negative control group and blank control group under normoxia. There was no significant difference in the expression of VEGF mRNA and MMP-2 mRNA between the three groups P> 0.05). The expression of HIF-1α mRNA, VEGF mRNA and MMP-2 mRNA in silencing group were lower than those in control group and blank control group at 6, 12 and 24 h hypoxia (P <0.05) There was no significant difference between the blank control group and the negative control group (P> 0.05). Compared with the normoxia group, the expressions of HIF-1α mRNA, VEGF mRNA and MMP-2 mRNA were significantly higher in the silenced group, the negative control group and the blank control group (P <0.05); Under hypoxia, the expression levels of HIF-1α mRNA, VEGF mRNA and MMP-2 mRNA in the three groups all showed a decreasing trend, and the differences among the three groups were statistically significant (P <0.05 ). Conclusions The expression of HIF-1α in human hepatocellular carcinoma SMMC-7721 cells is effectively silenced by RNAi technology, and the expression of VEGF mRNA and MMP-2 mRNA are decreased, suggesting that silencing HIF-1α gene can inhibit the invasion and metastasis of primary hepatocellular carcinoma .