靶向沉默BMI-1表达对食管癌细胞放射增敏实验研究

Effect of specific knockdown of BMI-1 on radiosensitivity enhancement in esophageal carcinoma cells

目的 采用siRNA干扰技术降低食管癌ECA109细胞BMI-1基因表达,观察其对放射线照射后细胞增殖、迁移能力及周期和凋亡影响.方法 针对BMI-1 mRNA序列,设计合成有效的干扰序列命名为BMI-1 siRNA组,阴性对照序列命名为NC组,未转染组命名为对照组.采用RT-PCR和蛋白印迹法检测ECA109中BMI-1在mRNA和蛋白水平的表达;Transwell小室实验检测siRNA干扰BMI-1基因对ECA109迁移能力的影响;MTT、流式细胞术和克隆形成实验检测BMI-1对ECA109放射敏感性的研究.结果 照射后BMI-1 siRNA组BMI-1基因mRNA和蛋白水平显著低于对照组和空载组,且细胞增殖和迁移能力显著降低（P=0.024、0.000、0.025、0.031）.克隆形成实验显示对照组、NC组放射敏感性相近（P=0.569）,而BMI-1 siRNA组细胞放射敏感性高于对照组和空载组（P=0.000）.流式细胞术分析显示照射后,BMI-1 siRNA组G2＋M期显著低于对照组和空载组（P=0.000）;且细胞凋亡显著增加（P=0.000）,而未照射组之间未见明显差异（P=0.350）.结论 siRNA干扰联合X线照射有效降低食管癌细胞ECA109中BMI-1基因表达,进而抑制亚致死性损伤修复而增加放射致死效率.

Objective To inhibit the expression of B-cell-specificmiv integration site-1（ BMI-1） in esophageal cancer ECA109 cells by siRNA interference, and to observe the effects of BMI-1 knockdown on cell proliferation, migration, cell cycle, and apoptosis after exposure to radiation. Methods Effective BMI-1 siRNA was designed and synthesized based on the sequence of the BMI-1 mRNA. ECA109 cells transfected with BMI-1 siRNA and negative control （ NC） siRNA were assigned to BMI-1 siRNA group and NC group, while ECA109 cells without transfection were set as a control. Real-time PCR and Western blot were used to determine the mRNA and protein expression of BMI-1 in ECA109 cells, respectively. The Transwell chamber assay was used to evaluate the migration ability of BMI-1-knockdown ECA109 cells. The MTT assay, flow cytometry, and colony formation assay were used to evaluate the effects of BMI-1 knockdown on the radiosensitivity of ECA109 cells. Results Compared with the NC group and the control group, the BMI-1 siRNA group had significantly lower mRNA and protein expression of BMI-1 and significantly reduced cell proliferation and migration after exposure （P=0. 024,P=0. 000）. According to the results of the colony formation assay, there was no significant difference in radiosensitivity between the control group and the NC group （ P=0. 025,P=0. 031） , while the BMI-1 siRNA group had significantly higher radiosensitivity than the control group and the NC group （P=0. 000）. According to the results of flow cytometry, the BMI-1 siRNA group had a significantly lower percentage of G2/M cells and significantly increased apoptosis after exposure than the control group and the NC group （ P=0. 000,0. 000）;however, there was no significant difference in apoptosis between the three groups before radiation （ P= 0. 350 ） . Conclusions SiRNA-mediated BMI-1 knockdown and X-ray radiation effectively reduce the expression of BMI-1, inhibit sublethal damage repair, and increase the radiation lethality in esophageal cancer ECA109 cells.