敲低Bmi-1对鼻咽癌细胞CNE2放射敏感性的影响

目的探讨敲低Bmi-1的表达水平对鼻咽癌细胞CNE2放射敏感性的影响。方法通过实时荧光定量PCR(qRT-PCR)和Western blotting检测4种鼻咽癌细胞株CNE1、CNE2、HNE1和HONE1中Bmi-1的表达,筛选出高表达Bmi-1的细胞株。设计3条针对Bmi-1 mRNA的干扰序列(shRNA1、shRNA2、shRNA3),用于构建慢病毒重组载体Bmi-1-shRNA1、Bmi-1-shRNA2、Bmi-1-shRNA3,然后转染至高表达Bmi-1的细胞株中,筛选出敲低Bmi-1效果最佳的慢病毒重组载体。将筛选出的慢病毒重组载体转染至高表达Bmi-1的鼻咽癌细胞株中,使用2 Gy放射线(IR)进行照射,通过平板克隆形成实验检测细胞增殖情况,流式细胞术检测细胞周期分布及凋亡情况。结果在4种鼻咽癌细胞株中,CNE2细胞的Bmi-1 mRNA表达水平最高(P<0.05),CNE1、CNE2细胞中Bmi-1蛋白表达相对较高(P<0.05)。构建的3条慢病毒重组载体Bmi-1-shRNA1、Bmi-1-shRNA2、Bmi-1-shRNA3均可降低CNE2细胞中Bmi-1的表达,且Bmi-1-shRNA3组最低(P<0.05)。在接受放射线照射后,与对照组(Bmi-1-NC+IR)相比,转染组(Bmi-1-shRNA3+IR)细胞增殖能力减弱克隆细胞数目:(130.67±12.01)个vs.(61.00±8.19)个],细胞凋亡率增加(13.40±0.66)%vs.(22.67±1.25)%],G₂/M期细胞比例增加(16.18±1.43)%vs.(22.34±2.00)%],差异有统计学意义(P均<0.05)。结论敲低Bmi-1表达可提高鼻咽癌CNE2细胞的放射敏感性。

Effects of Bmi-1 knockdown on radiosensitivity in nasopharyngeal carcinoma cell line CNE2

Objective To investigte the relationship between Bmi-1 knockdown and radiosensitivity of nasopharyngeal carcinoma cell CNE2 after irradiation.Methods The expression of Bmi-1 in nasopharyngeal carcinoma cells CNE1,CNE2,HNE1 and HONE1 was detected by quantiative real-time PCR(qRT-PCR)and Western blotting,and the cell lines with high expression of Bmi-1 were screened.Three interfering sequences(shRNA1,shRNA2,shRNA3)targeting Bmi-1 mRNA were designed to construct lentiviral recombinant vectors named Bmi-1-shRNA1,Bmi-1-shRNA2 and Bmi-1-shRNA3,and then transfected into cell lines with high expression of Bmi-1.The lentiviral recombinant vector with the best effect of knocking down Bmi-1 was screened.The selected lentivirus recombinant vector was transfected into nasopharyngeal carcinoma CNE2 cell with high expression of Bmi-1,and then irradiated with 2 Gy radiation(IR).The proliferation was detected by plate clone formation experiment,and the cell cycle distribution and apoptosis were detected by flow cytometry.Results Among the four nasopharyngeal carcinoma cell lines,the expression level of Bmi-1 mRNA in CNE2 cell was the highest(P<0.05),and the expression of Bmi-1 protein in CNE1 and CNE2 cells was relatively high(P<0.05).The constructed lentivirus recombinant vector reduced the expression of Bmi-1 in CNE2 cell,and the expression of Bmi-1 in Bmi-1-shRNA3 group was the lowest(P<0.05).After radiation exposure,compared with the control group(Bmi-1-NC+IR),the proliferation of CNE2 cell in the transfection group(Bmi-1-shRNA3+IR)decreased(the number of cloned cells:130.67±12.01 vs.61.00±8.19),the apoptosis rates increased(13.40±0.66)%vs.(22.67±1.25)%],and the proportion of G₂/M phase increased(16.18±1.43)%vs.(22.34±2.00)%],and these differences were statistically significant(P<0.05).Conclusion Knockdown of Bmi-1 expression can improve the radiosensitivity of nasopharyngeal carcinoma cell line CNE2.