Bmi-1通过抑制p16基因的表达促进人胰腺癌细胞的增殖

B-cell specific moloney leukemia virus insertion site 1 promotes proliferation of pancreatic cancer cells PANC-1 via inhibiting the expression of p16 gene

目的观察靶向下调Bmi-1基因对人胰腺癌细胞（PANC-1）细胞增殖、p16启动子甲基化、p16表达的影响。方法构建反义Bmi-1真核表达载体转染人人胰腺癌PANC-1细胞，运用荧光倒置显微镜观察、Western blot技术检测靶基因沉默效率；运用流式细胞术检测细胞周期及凋亡；Western blot、实时定量聚合酶链反应（Real-timePCR）检测p16基因表达；甲基化特异性聚合酶链反应（MSP）检测p16启动子甲基化变化。结果荧光倒置显微镜观察转染后48h细胞转染效率在90％左右；细胞周期出现阻滞（Go／G，期细胞由40．52％上升至60．48％，P〈0．05）、凋亡增加（由7．87％上升至21．67％，P〈0．05）；Bmi-1蛋白表达水平明显下降[吸光度（IA）值318．54±1．21到175．39±0．73，P〈0．05]；p16启动子甲基化水平降低而p16基因RNA（从3．563±0．128上升到8．621±0．310，P〈0．05）及蛋白质表达上升（IA值213．38±0．54到304．12±0．76，P〈0．05）。结论本实验构建的反义Bmi-1载体能够有效沉默细胞中靶基因的表达，抑制细胞增殖；Bmi-1很可能通过甲基化的方式对p16的表达进行调控。

Objective To study the effect of knocking down B-cell specific moloney leukemia virus insertion site 1 （ Bmi-1 ） on cell proliferation, the methylation of p16 and the expression of p16. Methods Antisense Bmi-1 vector was constructed and transfeeted into human pancreatic cancer cell line （ PANC-1 ） cells, and the efficiency of transfection was evaluated by fluorescence microscope and Western blotting. Cell cycle and apoptosis were examined by using flow cytometry. The expression of p16 and the methylation of p16 promotor were detected by using Western blotting, real-time polymerase chain reaction （PCR） and methylation-speeific PCR, respectively. Results Antisense Bmi-1 vector was successfully constructed, and the transfection efficiency was about 90%. The percentage of GOG1 cells was increased from 40. 52% to 60.48% （P 〈0. 05） and apoptosis rate increased from 7.87% to 21.67% （P 〈0. 05）. The expression of Bmi-1 protein was reduced from 318.54 ± 1.21 to 175.39 ±0. 73 （P 〈0. 05 ）, the expression levels of p16 RNA and protein in the transfected cells were increased to 8. 621± 0. 310 and 304. 12±0. 76 respectively （P 〈0. 05）, and the promotor methylation of p16 was declined （P 〈0. 05）. Conclusion Antisense Bmi-1 vector may knock down the expression of Bmi-1 in PANC-1 ceils and could inhibit the proliferation of cells. The expression of p16 is possibly regulated by Bmi-1 via promoting the methylation of p16 promotor.