过表达BMI-1对HeLa细胞中HOX基因表达和细胞周期的影响

目的： 将构建成功的真核表达载体pEGFP-BMI-1转染宫颈癌细胞系HeLa，检测其对同源盒（HOX）基因表达和细胞周期的影响。方法: 采用脂质体转染法，将质粒pEGFP-BMI-1 DNA瞬时转染HeLa细胞，确定融合蛋白B细胞特异性莫洛尼氏白血病毒插入位点1-加强型绿色荧光蛋白（BMI-1-EGFP）表达后，实时荧光定量RT-PCR方法检测转染前后HeLa细胞中周期素依赖性激酶抑制剂P16INK4a、人类端粒酶逆转录酶（hTERT）、同源盒A9（HOXA9）、同源盒B4(HOXB4)和同源盒C13(HOXC13) mRNA的表达变化，PI染色流式细胞仪检测细胞周期。结果: (1)在HeLa细胞中过表达BMI-1显著下调P16INK4a、HOXA9和HOXC13 mRNA的表达，分别平均降低为对照组的9.2%、10.9%和69.7%(P<0.01)，而hTERT和HOXB4 mRNA的表达变化无显著差异(P＞0.05)。(2)pEGFP-BMI-1转染HeLa细胞后，G1期细胞由65.68％减少至50.53％，S期细胞则由27.17％增加至39.59％(P<0.01)。结论: 真核表达载体pEGFP-BMI-1转染HeLa细胞过表达外源性BMI-1，显著下调P16INK4a、HOXA9和HOXC13的表达，同时G1期细胞减少、S期细胞增加，这可能是BMI-1参与肿瘤发生发展的机制之一。

Effects of BMI-1 over-expression on HOX family expression and cell cycle in HeLa cells

AIM: To observe whether transfection of mammalian expression vector pEGFP containing the gene of B-cell specific moloney leukemia virus insertion site 1(BMI-1) could express in human cervix cancer cell line HeLa, and to detect its effect on HOX family expression and cell cycle.METHODS: pEGFP-BMI-1 was transfected into HeLa cells with Lipofectamine 2000. The expression of pEGFP-BMI-1 was determined by EGFP fluorescence and Western blotting. SYBR green I real-time RT-PCR was used to quantitate mRNA expression of P16INK4a, hTERT, HOXA9, HOXB4 and HOXC13. FACS analysis was used to detect the change of cell cycle.RESULTS: In HeLa cells transfected with pEGFP-BMI-1, the results of real-time RT-PCR showed that the mRNA expressions of P16INK4a, HOXA9 and HOXC13 were reduced to 9.2%, 10.9% and 69.7%, respectively, as compared to control HeLa cells (P<0.01). However, hTERT and HOXB4 mRNA expressions did not change significantly (P>0.05). FACS analysis showed a decrease from 65.68 % to 50.53% in G1 population and a significant increase from 27.17% to 39.59 % in S population after transfection (P<0.01).CONCLUSION: BMI-1 over-expression in HeLa cells down-regulates mRNA expressions of P16INK4a, HOXA9 and HOXC13, decreases G1 population and increases S population. Therefore, BMI-1 may be involved in carcinogenesis and cancer development.