SET8 siRNA对食管癌TE1细胞周期以及化疗敏感性的影响

Effects of SET8 siRNA on cell cycle and chemosensitivity of TE1 cells

目的食管癌是最常见的恶性肿瘤之一,目前对食管癌发生发展相关分子机制的研究成为热点,寻找新的肿瘤分子标志物,以期提高食管癌的早期诊治水平。本研究探讨siRNA沉默SET8基因对食管癌TE1细胞周期、细胞凋亡和药物敏感性的影响。方法设计并体外转录合成针对人SET8基因的特异性siRNA,并通过脂质体(Lipofectamine^(TM) 2000)将SET8 siRNA转染入TE1细胞。蛋白质印迹法检测转染前后TE1细胞中SET8蛋白表达的变化,流式细胞术分析SET8siRNA对TE1细胞周期及细胞凋亡的影响,MTS法观察SET8基因在TE1细胞中表达沉默对紫杉醇、顺铂、5-FU、依托泊苷敏感性的影响。结果 SET8-siRNA转染组抑制了SET8蛋白表达水平,与两对照组相比分别降低了79.70%。SET8表达沉默可导致细胞周期障碍,细胞周期于SET8-siRNA转染后72h G_2/M期阻滞(P<0.01),并诱导TE1细胞凋亡,P<0.01。紫杉醇处理后,SET8-siRNA组和阴性对照组的半抑制率浓度(IC_(50))分别为(66.85±0.64)和(117.24±3.18)μg/L,SET8-siRNA转染食管癌细胞株TE1对紫杉醇药物更加敏感(t=-26.906,P=0.001),而顺铂、依托泊苷和5-FU处理后,SET8-siRNA组和阴性对照组的IC_(50)差异无统计学意义,均P>0.05。结论靶向SET8-siRNA能明显下调靶基因SET8基因的表达,导致细胞周期障碍,诱导细胞凋亡,并能提高食管癌对紫杉醇的敏感性,为食管癌的治疗提供新的策略。

OBJECTIVE To investigate the influence of SET8 gene silencing by RNA interference on cell cycle,apoptosis and drug sensitivity of TE1 cells.METHODS Designed and transcriptive synthesized fluorescein-labeled siRNA targeting the SET8 gene in vitro were transfected into TE1 cells by Lipofectamine 2000,the expressions of SET8 protein in esophagus carcinoma cells were evaluated by western blotting,and the cell cycle and apoptosis was examined by FCM,the chemsensitivity of transfected cell to paclitaxel,cisplatin,5-fluorouracil and etoposide was determined by MTS assay.RESULTS SET8 siRNA significantly downregulated the expression of SET8 gene in esophagus carcinoma cell at protein level 79.70%.The expression of SET8 downregulated resulted in disorder of the cell cycle,and the cell cycle was arrested in G2/M phase after SET8-siRNA transfection for 72 h(P<0.01),and SET8 siRNA induced obvious apoptosis of TE1 cell(P<0.01).The IC50 of groups of SET8 siRNA and negative siRNA treated with paclitaxel were(66.85±0.64)and(117.24±3.18)μg/L.The SET8 siRNA transfected TE1 cells were much more sensitive to paclitaxel compared to the negative siRNA transfected TE1 cells(P<0.01),but the IC50 of groups of SET8 siRNA and negative siRNA treated with cisplatin,5-fluorouracil and etoposide had no obvious difference and statistically significant.CONCLUSIONS The effective siRNA targeting SET8 can down-regulate SET8 gene expression and cause disorder of the cell cycle,induce obvious apoptosis of TE1 cell,and enhance the drug sensitivity of the TE1 cells to paclitaxel.It is expected to provide a novel therapy approach for esophageal cancer.