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目的探讨RNA干扰乏氧诱导因子-1α(HIF-1α)基因对CoCl2诱导的乏氧人肝癌细胞SMMC-7721放射敏感性的影响及其机制。方法利用分子生物学技术构建靶向HIF-1α基因的RNA干扰质粒pGenesil-HIF,脂质体介导转染SMMC-7721细胞后进行乏氧培养,分别采用RT-PCR和Western blot法检测HIF-1α基因的mRNA和蛋白表达,四甲基偶氮唑盐(MTT)法检测细胞增殖活性,流式细胞术检测细胞周期和细胞凋亡,克隆存活实验检测细胞放射敏感性的变化。结果酶切鉴定和测序证实重组质粒pGenesil-HIF构建正确,转染质粒后乏氧培养24 h,SMMC-7721细胞HIF-1α基因的mRNA和蛋白表达水平明显低于对照组和阴性干扰组(P<0.05或P<0.01);乏氧培养24 h、48 h和72 h后,HIF-1α干扰组细胞增殖活性明显低于对照组(P<0.05或P<0.01),细胞阻滞于G0/G1期(P<0.01);乏氧培养48 h和72 h后HIF-1α干扰组细胞凋亡百分率明显高于对照组(P<0.01);乏氧培养72 h后HIF-1α干扰组SMMC-7721细胞放射增敏比为1.45。结论 RNA干扰HIF-1α基因可增强乏氧人肝癌细胞SMMC-7721的放射敏感性,其机制可能与RNA干扰HIF-1α基因抑制乏氧细胞增殖和诱导凋亡有关。
Objective To investigate the effects of HIF-1α RNA interference on the radiosensitivity of CoCl2-induced hepatoma SMMC-7721 cells and its mechanism. Methods RNA interference plasmid pGenesil-HIF targeting HIF-1α gene was constructed by molecular biology technique. After lipofectamine was transfected into SMMC-7721 cells, hypoxia culture was performed. The expression of HIF-1α was detected by RT-PCR and Western blot respectively. 1α gene mRNA and protein expression, MTT assay cell proliferation, cell cycle and apoptosis were detected by flow cytometry, cell survival assay was used to detect changes in cell radiosensitivity. Results The recombinant plasmid pGenesil-HIF was constructed correctly by restriction enzyme digestion and sequencing. The expression of HIF-1α mRNA and protein in SMMC-7721 cells was significantly lower than that in the control and negative control groups (P <0.05 or P <0.01). After hypoxic culture for 24 h, 48 h and 72 h, the cell proliferation of HIF-1α interference group was significantly lower than that of the control group (P <0.05 or P <0.01) G1 phase (P <0.01). The percentage of apoptosis of HIF-1α interference group after hypoxia for 48 h and 72 h was significantly higher than that of control group (P <0.01) 7721 cells radiosensitivity ratio of 1.45. Conclusion RNA interference of HIF-1α gene enhances the radiosensitivity of hypoxic human hepatocellular carcinoma cell line SMMC-7721, which may be related to the inhibition of HIF-1α gene proliferation and apoptosis induced by hypoxia.