Effect of siRNA-mediated silencing Bmi-1 gene expression on the proliferation of lung cancer cell line A549<em>in vitro</em>and<em>in vivo</em>

郑翔宇; 朱杰; 王艺芳; 刘纯青; 刘奔; 杨春辉; 刘丹丹; 孟秀香

doi:10.3969/j.issn.1007-3969.2013.07.005

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Effect of siRNA-mediated silencing Bmi-1 gene expression on the proliferation of lung cancer cell line A549in vitroandin vivo

更新时间：2025-12-31

- Effect of siRNA-mediated silencing Bmi-1 gene expression on the proliferation of lung cancer cell line A549in vitroandin vivo
- China Oncology Vol. 23, Issue 7, Pages: 505-511(2013)
- 作者机构：
  
  1. 大连医科大学检验医学院,辽宁,大连,116044
  2. 河南省中医院检验科,河南,郑州,450002
  3. 大连医科大学第二临床学院检验科,辽宁,大连,116027
- 作者简介：
- 基金信息：
- DOI：10.3969/j.issn.1007-3969.2013.07.005
  CLC：
- Published Online：03 March 2014，
  
  Published：2013
- 稿件说明：
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郑翔宇,朱杰,王艺芳,刘纯青,刘奔,杨春辉,刘丹丹,孟秀香. Bmi-1-siRNA对肺腺癌A549细胞体内外增殖能力的影响[J]. 中国癌症杂志, 2013, 23(7): 505-511.

郑翔宇, 朱杰, 王艺芳, et al. Effect of siRNA-mediated silencing Bmi-1 gene expression on the proliferation of lung cancer cell line A549in vitroandin vivo[J]. China Oncology, 2013, 23(7): 505-511.
郑翔宇,朱杰,王艺芳,刘纯青,刘奔,杨春辉,刘丹丹,孟秀香. Bmi-1-siRNA对肺腺癌A549细胞体内外增殖能力的影响[J]. 中国癌症杂志, 2013, 23(7): 505-511. DOI： 10.3969/j.issn.1007-3969.2013.07.005.

郑翔宇, 朱杰, 王艺芳, et al. Effect of siRNA-mediated silencing Bmi-1 gene expression on the proliferation of lung cancer cell line A549in vitroandin vivo[J]. China Oncology, 2013, 23(7): 505-511. DOI： 10.3969/j.issn.1007-3969.2013.07.005.

摘要

背景与目的：原癌基因Bmi-1是多梳基因家族中的一员，能调节正常干细胞和肿瘤干细胞的自我更新能力。近年来发现其在多种恶性肿瘤中表达上调。本文旨在观察Bmi-1基因沉默对肺腺癌A549细胞体内外增殖的影响，并初步探讨其机制。方法：根据本实验室设计的4条针对Bmi-1的小干扰RNA(siRNA)序列，选择一条已经证实最有效的序列作为靶序列和一条随机序列作为阴性对照，构建重组逆转录病毒siRNA表达载体并将其转染入A549细胞中；应用RT-PCR和蛋白质印迹法(Western blot)检测对Bmi-1基因的沉默效果；应用MTT比色法、台盼蓝拒染法及平板克隆形成实验检测Bmi-1-siRNA对A549细胞体外增殖的影响；利用流式细胞仪分析各组细胞的细胞周期；通过裸鼠腋窝皮下接种各组细胞，观察Bmi-1-siRNA对A549细胞在裸鼠体内的致瘤能力的影响；Western blot检测PTEN、p-AKT、cyclin D1、P21、P27蛋白表达。结果：Bmi-1-siRNA有效地沉默了Bmi-1基因mRNA和蛋白的表达；沉默Bmi-1基因的表达能够抑制A549细胞的体内外增殖能力，使干扰组细胞的细胞周期阻滞于G1期；沉默Bmi-1基因的表达后，干扰组细胞中PTEN、P21、P27蛋白增加，p-AKT、cyclin D1蛋白表达降低。结论：Bmi-1-siRNA通过使细胞周期阻滞于G1期来抑制肺腺癌A549的体内外增殖能力，这种抑制作用涉及cyclin D1和p-AKT表达下降以及P21/P27和PTEN的表达上调。

Abstract

Background and purpose: The pro-oncogene Bmi-1 is a member of the polycomb- group family

can regulation of the proliferation and self-renewal of normal and tumor stem cells. In recent years

Bmi-1 has been found that it is overexpressed in varieties of human malignant tumors. The study aimed to observe the effects of Bmi-1-siRNA on the growth capacity of lung cancer cell line A549 in vivo and in vivo

and explore its mechanism. Methods: The most effective one as a target sequence was chosen from four Bmi-1 siRNA sequences which were designed by our lab

and one random sequence was chosen as a negative control. In short

the chemically synthesized siRNA and control sequences were connected to a retrovirus expressing vector

pSUPERretro-Neo plasmid

and then transfected into A549 cells. The stably transfected cells were cultured and passed. The level of mRNA and protein of Bmi-1 in A549 cells were assessed by RT-PCR and Western blot respectively. The proliferations of A549 cells in vivo was analyzed with MTT

trypan blue exclusion and plate colony forming methods. Flow cytometry was used for cell cycle analysis. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of A549 cells. The expressions of cyclin D

p21/27

p-AKT and PTEN were analyzed by Western blot. Results: Compared to A549-ctr and A549-wt cells

Bmi-1 mRNA and protein levels all significantly reduced in A549-Bmi-1-siRNA cells. Bmi-1-siRNA inhibited the growth

colony formation in vitro and tumorigenesis in vitro of A549 cells

and the interference cells cell cycle arrested in G1 phase. In A549-Bmi-1-siRNA cells

p-AKT and cyclinD1 expression were down-regulated while p21/p27 and PTEN were up-regulated. Conclusion: Silencing Bmi-1 gene inhibits the proliferation of A549 cells through G1 phase arrest

which involves the downregulation of cyclin D/p-AKT and upregulation of p21/p27/PTEN.

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Effect of siRNA-mediated silencing Bmi-1 gene expression on the proliferation of lung cancer cell line A549in vitroandin vivo

DOI：10.3969/j.issn.1007-3969.2013.07.005

摘要

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