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目的利用小干扰RNA(small interfering RNA,si RNA)片段沉默卵巢癌细胞人垂体瘤转化基因1(human pituitary tumor-transforming gene 1,h PTTG1)基因表达,探讨其抑制细胞凋亡的分子机制。方法通过脂质体将h PTTG1 si RNA转染A2780细胞(h PTTG1 si RNA干扰组),并设立正常组和阴性对照组,转染48 h后进行检测。采用实时荧光定量聚合酶链反应检测转染前后h PTTG1 m RNA表达水平的变化,采用逆转录-聚合酶链反应和蛋白质印迹法检测survivin m RNA和蛋白表达水平,采用DNA梯带电泳法和碘化丙啶单染法分析细胞凋亡,采用比色法测定胱天蛋白酶(caspase)-3活性。结果 h PTTG1 si RNA可抑制A2780细胞内h PTTG1 m RNA表达;h PTTG1 si RNA干扰组可见典型的细胞凋亡阶梯状电泳,流式细胞仪检测该组细胞凋亡率为(17.53±2.17)%,明显高于正常组和阴性对照组[(8.97±1.56)%、(9.64±1.31)%],差异有统计学意义(P<0.05);h PTTG1干扰后survivin m RNA和蛋白表达均下调,caspase-3活性增强。结论 h PTTG1 si RNA可下调survivin基因表达,活化caspase-3,导致A2780细胞凋亡,其可成为卵巢癌基因治疗的潜在靶点。
OBJECTIVE: To study the molecular mechanism of hPTTG1 gene silencing in human ovarian cancer cell line HpTG1 by using small interfering RNA (siRNA). Methods h PTTG1 si RNA was transfected into A2780 cells by liposomes (h PTTG1 si RNA interference group), and normal control group and negative control group were established. The expression of hTTG1 m RNA before and after transfection was detected by real-time fluorescence quantitative polymerase chain reaction. The expression of m RNA and protein of survivin was detected by reverse transcription-polymerase chain reaction and Western blotting. DNA ladder electrophoresis and iodine Apoptosis was analyzed by propidium iodide staining and the activity of caspase-3 was measured by colorimetry. Results h PTTG1 si RNA inhibited the expression of h PTTG1 m RNA in A2780 cells. H A typical apoptotic ladder electrophoresis was observed in PTTG1 si RNA interference group. The apoptotic rate was (17.53 ± 2.17)% in flow cytometry (8.97 ± 1.56)%, (9.64 ± 1.31)% respectively], the difference was statistically significant (P <0.05); h The expression of survivin m RNA and protein were down-regulated after PTTG1 interference, caspase-3 activity increased. Conclusion h PTTG1 si RNA can down-regulate the expression of survivin gene and activate caspase-3, resulting in apoptosis of A2780 cells. It may be a potential target for gene therapy of ovarian cancer.