mRNA干扰BMI-1对人食管癌细胞放射增敏作用研究

Effect of BMI-1 on radiosensitization of esophageal carcino-ma cells after silencing of BMI-1 gene

目的 研究BMI-1对食管癌TE-13细胞放射敏感性的影响及其机制。方法 以TE-13细胞为研究对象,分为转染有效BMI-1 mRNA干扰序列（siRNA）即BMI-1 siRNA组、转染阴性对照序列即NC组、未转染即control组。qRT-PCR和蛋白印迹法检测TE-13细胞中BMI-1 mRNA和蛋白表达水平;MTT法和克隆形成实验检测BMI-1对食管癌TE-13细胞增殖和放射敏感性影响;流式细胞术检测BMI-1对TE-13细胞周期、凋亡的影响;蛋白印迹法检测细胞中Bcl-2、Bax蛋白表达水平。组间差异采用方差分析。结果 BMI-1 siRNA组BMI-1 mRNA和蛋白表达水平均显著低于control、NC组（P=0.000、0.000）。照射后BMI-1 siRNA组肿瘤细胞的增殖水平、克隆形成能力均显著下降（P=0.031、0.000）;照射后BMI-1 siRNA组G2＋M期细胞比例显著低于control、NC组（P=0.000、0.000）,且凋亡率明显增加（P=0.000、0.000）,同时Bcl-2的表达显著降低（P=0.000、0.000）,而Bax表达明显增加（P=0.000、0.000）。结论 干扰siRNA抑制了食管癌TE-13细胞中BMI-1基因的表达,消除了G2＋M期阻滞,显著提高了电离辐射后细胞凋亡水平,增加了放射敏感性,该作用可能与Bcl-2和Bax基因表达有关。

Objective To investigate the effects of BMI-1 expression inhibition by RNA interference on the radiosensitivity of esophageal cancer TE-13 cells and its mechanism. Methods The siRNA based on the sequence of BMI-1 mRNA was synthesized to transfect cultured TE-13 cells as BMI-1 siRNA group, a negative one was synthesized to transfect cultured TE-13 cells as negative control group （NC group）, and untransfected TE-13 cells were named as control group. The expression of the BMI-1 mRNA and protein in TE-13 cells was measured by quantitative real-time PCR and Western blot, respectively. The cell proliferation and the radiosensitivity of TE-13 cells were measured by MTS and colony-forming assay, respectively. Flow cytometry was used to analyze cell cycle and apoptosis. The expression of BCL-2 and BAX in TE-13 cells was measured by Western blot. Comparison between groups was made by analysis of variance. Results The BMI-1 siRNA group had significantly lower expression of BMI-1 mRNA and protein than the control group and the NC group （P=0.000,0.000）. The proliferation of TE-13 cells in the BMI-1 siRNA group decreased significantly after irradiation （P=0.031）. The colony-forming assay showed that the BMI-1 siRNA group had a significantly higher radiosensitivity than the control group and the NC group （P=0.000）. After irradiation, the BMI-1 siRNA group had a significantly lower percentage of cells in G2/M phase than the control group and the NC group （P=0.000,0.000）. The BMI-1 siRNA group had a significantly increased apoptosis rate （P=0.000,0.000）,significantly reduced expression of BCL-2（P=0.000,0.000）, and significantly increased expression of BAX after irradiation （P=0.000,0.000）. Conclusions BMI-1 siRNA can inhibit the expression of BMI-1 gene in esophageal cancer TE-13 cells, eliminate the cell cycle arrest in G2/M phase, induce cell apoptosis after ionizing irradiation in vitro, and increase the radiosensitivity, which may be related to the regulation of the expression of BCL-2 and BAX.