mRNA干扰BMI-1对人食管癌细胞放射增敏作用研究

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。

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

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.