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目的观察缺氧诱导因子-1α(HIF-1α)特异性小分子干扰 RNA(siRNA)对肝癌化疗的增敏作用。方法构建 HIF-1αsiRNA 真核表达质粒,经脂质体稳定转染于耐药的 C28肝癌细胞。研究分为实验组、阴性对照组和空白对照组。荧光定量 PCR 和 Western blot 技术分别检测3组细胞中多药耐药相关基因 MDR1、LRP、MRP1在 mRNA 和蛋白水平的表达,PI 法流式细胞术分析3组细胞在受到5-Fu 作用后的凋亡指数。每组细胞分别随机注入10只裸鼠皮下,比较3组细胞成瘤后对 ADM 治疗的反应性。结果 HIF-1αsiRNA 能有效抑制 HIF-1α基因的表达,并能使 C28耐药肝癌细胞内 MDR1、MRP1、LRP 基因在 mRNA 和蛋白水平表达明显下降。在5-Fu 作用后第24、48、72小时,实验组细胞的凋亡指数分别是阴性对照组的2.88、3.56和3.87倍,实验组对化疗药物5-Fu 的敏感性显著增强(P<0.01)。注射阿霉素后,实验组裸鼠皮下的耐药肝癌瘤体明显缩小,肿瘤抑制率为41.35%,与阴性对照组比较差异有统计学意义(P<0.01)。结论成功构建了 HIF-1α-siRNA 的真核表达质粒。HIF-1α靶序列特异性的 siRNA 可增强肝癌细胞对化疗药物的敏感性,它与传统化疗药物的联合应用可望实现对肝癌的有效治疗。
Objective To observe the sensitizing effect of hypoxia inducible factor-1α (HIF-1α) -specific small interfering RNA (siRNA) to chemotherapy of hepatocellular carcinoma (HCC). Methods The eukaryotic expression plasmid of HIF-1α was constructed and stably transfected into drug-resistant C28 hepatoma cells by liposome. The study was divided into experimental group, negative control group and blank control group. The expression of multidrug resistance-related genes MDR1, LRP and MRP1 at mRNA and protein levels were detected by real-time quantitative PCR and Western blot respectively. The expressions of MDR1, LRP and MRP1 in 3 groups of cells were detected by PI-flow cytometry after 5-Fu treatment Apoptosis index. Each group of cells were randomly injected subcutaneously 10 nude mice, compared 3 groups of tumor response to ADM treatment after tumor formation. Results HIF-1α siRNA could effectively inhibit the expression of HIF-1α gene and significantly decrease the mRNA and protein expression of MDR1, MRP1 and LRP in C28 resistant hepatoma cells. After 24 hours, 48 hours and 72 hours after 5-Fu treatment, the apoptotic index in the experimental group was 2.88, 3.56 and 3.87 times higher than that in the negative control group, respectively. The sensitivity of the experimental group to chemotherapeutic drug 5-Fu was significantly increased (P < 0.01). After injection of doxorubicin, the experimental group of subcutaneous drug-resistant liver cancer in nude mice significantly reduced, the tumor inhibition rate was 41.35%, compared with the negative control group, the difference was statistically significant (P <0.01). Conclusion The eukaryotic expression plasmid of HIF-1α-siRNA was successfully constructed. HIF-1α target-specific siRNA can enhance the sensitivity of hepatoma cells to chemotherapeutic drugs. Combined with traditional chemotherapeutic drugs, it is expected to be effective in the treatment of liver cancer.