敲减BMI-1表达诱导HeLa细胞G_1期阻滞  

Knock-down of BMI-1 Arrests HeLa Cells in G_1 Phase

作　　者：陈凤花[1] 李一荣[1] 王琳[1] 胡丽华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院检验科,武汉430022

出　　处：《中国生物化学与分子生物学报》2010年第2期134-139,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：湖北省科技攻关项目(No.2005AA304B08)~~

摘　　要：B细胞特异性莫洛尼氏白血病毒插入位点1(B-cell specific moloney leukemia virus insertionsite1,BMI-1)在多种恶性肿瘤中高表达.为探索BMI-1在宫颈癌发生发展过程中的生物学作用,将构建成功并已鉴定为有效序列的短发夹状RNA(short hairpin RNA,shRNA)真核表达载体BMI-1shRNA2和BMI-1shRNA4,转染人宫颈癌细胞系HeLa并筛选稳定转染细胞株,Western印迹检测BMI-1蛋白表达,实时荧光定量RT-PCR检测周期素依赖性激酶抑制剂p16INK4a(cyclin-dependentkinase inhibitor p16INK4a)、人类端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)、同源盒A9(homeoboxA9,HOXA9)、同源盒B4(homeoboxB4,HOXB4)和同源盒C13(homeoboxC13,HOXC13)基因mRNA的表达变化,PI染色流式细胞仪检测细胞周期变化.结果表明,在稳定转染BMI-1shRNA2、BMI-1shRNA4的HeLa细胞中,显著抑制BMI-1蛋白表达,上调p16INK4a、HOXA9和HOXC13三者mRNA表达,而hTERT和HOXB4两者mRNA的表达水平无明显改变,同时G1期细胞增加、S期细胞减少,提示敲减BMI-1表达诱导HeLa细胞G1期阻滞,BMI-1可能是宫颈癌基因治疗的靶分子.The over-expression of B-cell specific moloney leukemia virus insertion site 1（BMI-1） is associated with human malignancies.To explore the effect of BMI-1 on cervical carcinogenesis,the short hairpin RNA（shRNA） eukaryotic expression vectors including BMI-1 shRNA2 and BMI-1 shRNA4 constructed successfully and identified efficiently were transfected into human cervical adenocarcinoma HeLa cells using lipofectamine 2000 and screened by G418.BMI-1 protein expression was analyzed by Western blotting,the mRNA expression levels of cyclin-dependent kinase inhibitor p16 INK4a,human telomerase reverse transcriptase（hTERT）,homeobox A9（HOXA9）,homeobox B4（HOXB4） and homeobox C13（HOXC13） were evaluated by SYBR greenⅠreal-time RT-PCR,and cell cycle was determined by FACS analysis.The results showed that BMI-1 protein expression was suppressed,and BMI-1 shRNA2 and BMI-1 shRNA4 significantly up-regulated the mRNA expression of p16 INK4a,HOXA9 and HOXC13,but had no effect on the mRNA expression of hTERT and HOXB4.FACS analysis showed a significant increase in G_1 population and an obvious decrease in S population.Our data suggested that RNA interference（RNAi）-mediated knock-down of BMI-1 could induce G_1 phase cell cycle arrest and up-regulate p16 INK4a,HOXA9 and HOXC13 expression in HeLa cells,and targeting BMI-1 might be a therapeutic potential for the treatment of cervical cancer.

关 键 词：B细胞特异性莫洛尼氏白血病毒插入位点1(BMI-1) 短发夹状RNA(shRNA) 周期素依赖性激酶抑制剂p16INK4a 同源盒A9 同源盒C13 

分 类 号：Q291[生物学—细胞生物学] Q786