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目的:探讨质粒介导RNA干扰抑制HIF-1α基因对肺癌A549细胞转移和侵袭能力的影响。方法:利用基因工程方法构建HIF-1α的干扰表达载体,并将其转染至肺腺癌A549细胞;分别用RT-PCR方法、Western blot方法检测细胞转染前后HIF-1α以及MMP-2的mRNA及蛋白表达的变化。细胞划痕试验和Transwell实验检测转染前后肺腺癌A549细胞迁移及侵袭能力的改变。结果:转染后肺腺癌A549细胞中HIF-1α和MMP-2的mRNA表达分别下降86%和64%(P<0.05)。转染后细胞蛋白表达明显下降。转染前后穿膜细胞数分别为(135.2±13.2)、(63.7±10.5),差异具有统计学意义(P<0.05)。转染前后划痕宽度分别为(0.48±0.14)、(1.04±0.15)mm,差异具有统计学意义(P<0.05)。结论:RNAi可有效抑制HIF-1α的表达,同时可结合MMP-2启动子下调MMP-2基因和蛋白的表达,降低肺癌A549细胞的转移和侵袭能力。
Objective: To investigate the effect of plasmid-mediated RNA interference (SRAP) on HIF-1α gene transfer and invasion in lung cancer A549 cells. METHODS: The expression vector of HIF-1α was constructed by gene engineering and transfected into lung adenocarcinoma A549 cells. The HIF-1α and MMP-2 were detected by RT-PCR and Western blot respectively. Changes in mRNA and protein expression. The cell scratch test and transwell assay were used to detect the migration and invasion of lung adenocarcinoma A549 cells before and after transfection. Results: The mRNA expression of HIF-1α and MMP-2 in lung adenocarcinoma A549 cells after transfection decreased by 86% and 64%, respectively (P<0.05). After transfection, the expression of cell protein was significantly decreased. The number of perfused cells before and after transfection was (135.2±13.2), (63.7±10.5), and the difference was statistically significant (P<0.05). The widths of scratches before and after transfection were (0.48±0.14) and (1.04±0.15) mm, respectively, and the difference was statistically significant (P<0.05). Conclusion: RNAi can effectively inhibit the expression of HIF-1α, and it can also bind MMP-2 promoter to down-regulate the expression of MMP-2 gene and protein, and reduce the metastasis and invasion ability of lung cancer A549 cells.