干扰基因BMI-1表达对食管癌细胞放射敏感性的影响

Effect of RNAi targeting BMI-1 gene on radiosensitivity of esophageal carcinoma cells

背景与目的:B细胞特异性的莫洛尼白血病毒插入位点1(B-cell-specific Moloney leukemia virus insertion site 1,BMI-1)期在放疗后DNA损伤中发挥重要作用。该研究采用siRNA干扰技术降低食管癌TE13细胞中BMI-1基因表达,探讨BMI-1的表达对X射线照射后食管癌细胞放射敏感性的影响。方法:针对BMI-1 mRNA序列,3对有效的干扰序列(siRNA1、siRNA2和siRNA3)和阴性对照序列由公司设计合成,瞬时转染食管癌TE13细胞,命名为BMI-1 siRNA1、BMI-1 siRNA2和BMI-1 siRNA3共3组,转染阴性对照序列组命名为NC组,未转染组命名为对照组。采用反转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)和蛋白[质]印迹法(Western blot)检测TE13各组中BMI-1在mRNA和蛋白水平的表达。采用MTT法检测BMI-1基因转染对TE13细胞增殖能力的影响。克隆形成实验检测BMI-1对TE13放射敏感性的作用。流式细胞术测定siRNA干扰BMI-1基因对细胞周期和凋亡分布的影响。RT-PCR和Western blot检测细胞内p16、CDK4基因和蛋白表达水平。结果:3对干扰序列使人BMI-1基因在mRNA和蛋白表达水平低于对照组和NC组,尤以siRNA3组效果最明显。选择siRNA3作为干扰组进行MTT和流式细胞术。MTT结果表明,BMI-1 siRNA3转染后BMI-1的低表达对细胞的增殖能力无明显影响;克隆形成实验的结果显示,空白对照组、NC组、干扰组的D_0分别为2.514、2.506和1.761 Gy;D_q值分别为2.694、2.664和2.122 Gy;外推数N值分别为2.920、2.895和3.336;SF2值分别为0.826 8、0.823 1和0.625 5,可见对照组和空载组间放射敏感性差异无统计学意义(P>0.05),而干扰组细胞的放射敏感性高于对照组和NC组;流式细胞术分析显示,6 Gy照射后,BMI-1 siRNA组G_0/G_1期比例明显高于对照组和空载组,G_2/M期显著低于对照组和NC组,而未照射时BMI-1的低表达对细胞周期无明显影响。干扰BMI-1基因后TE13细胞p16基因和蛋白水平升高(P<0.01),CDK4基因和蛋白表达水平显著降低(P<0.01)。结论:siRNA干扰技术有效地抑制了食管癌细胞TE13中BMI-1基因的表达,联合X线照射后显著消除了细胞周期在G_2/M期的阻滞,增加了食管癌的放射敏感性,其诱导作用与p16和CDK4基因和蛋白表达有关。

Background and purpose： B cell-specific MLV integration site 1 （BMI-1） gene plays an important role in DNA damage after exposure to irradiation. The present study aimed to investigate the effect of BMI-1 on radiosensitivity of esophageal carcinoma cell after down-regulation of BMI-1 expression by silencing siRNA. Methods： Three pairs of siRNA based on the sequences of the BMI-1 mRNA were synthesized （siRNA1, siRNA2 and siRNA3） by compa-ny, and transfected into cultured TE13 cells as the BMI-1 siRNA groups, and a negative one was synthesized to be used as the negative control （NC） group. The untransfected group was named as the control group. BMI-1 mRNA and protein expression in esophageal cancer TE13 cells were detected by reverse transcription polymerase chain reaction （RT-PCR） and Western blot in different groups. This study used flow cytometry assay to analyze cell cycle of transfected cells, and examined cellular growth and radiosensitivity in vitro by MTT and clone formation assay. mRNA and protein expression of p16 and CDK4 in esophageal cancer TE13 cells were detected by RT-PCR and Western blot. Results： The results of RTPCR and Western blot showed that the expressions of BMI-1 at gene and protein levels were inhibited after silencing the BMI-1 gene. The mRNA and protein expression of BMI-1 in BMI-1 siRNA3 group were both significantly lower than that in BMI-1 siRNA1 and 2 groups. There was no significant difference in the cell proliferation among control, NC and BMI-1 siRNA3 groups. The values of D0, Dq, and SF2 in BMI-1 siRNA3 group were 1.761, 2.122 and 0.6255, respectively, obviously lower than those in control group （2.514, 2.694 and 0.8268） and those in NC group （2.506, 2.664 and 0.8231）, while the value of N in BMI-1 siRNA3 group （3.336） was higher than that in control group （2.92） and that in NC group （2.895）, which showed higher radiosensitivity in BMI-1 siRNA3 group. In addition, the cell cycle was arrested at G2/M phase after irradiation in control and NC groups. The percentage of G0/G1 phase in BMI-1 siRNA3 group was higher than that of control group and NC group, while the percentage of G2/M phase was lower than those in the latter. The up-regulation of p16 and down-regulation of CDK4 at gene and protein levels were detected after knockdown of BMI-1 expression by siRNA （P〈0.01）. Conclusion： siRNA could inhibit BMI-1 gene expression in esophageal cancer TE13 cells and enhance radiosensitivity, followed by eliminating the cell cycle arrest at G2/M stage after irradiation in vitro, which is related to the regulation of the protein expression of p16 and CDK4.