Bmi-1-siRNA对肺腺癌A549细胞体内外增殖能力的影响

doi:10.3969/j.issn.1007-3969.2013.07.005

中国癌症杂志 ›› 2013, Vol. 23 ›› Issue (7): 505-511.doi: 10.3969/j.issn.1007-3969.2013.07.005

Bmi-1-siRNA对肺腺癌A549细胞体内外增殖能力的影响

郑翔宇^1,2,朱杰³,王艺芳¹,刘纯青¹,刘奔¹,杨春辉¹,刘丹丹¹,孟秀香¹

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

出版日期:2013-07-25 发布日期:2014-03-03
通信作者: 孟秀香　E-mail:xiuxiang_meng@sina.com
基金资助:
辽宁省自然科学基金(No: 20072169)

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

ZHENG Xiang-yu^1,2,ZHU Jie³,WANG Yi-fang¹,LIU Chun-qing¹,LIU Ben¹,YANG Chun-hui¹,LIU Dan-dan¹,MENG Xiu-xiang¹

1.College of Laboratory Medicine of Dalian Medical University, Dalian Liaoning 116044, China;
2. Department of Laboratory Diagnosis, Henan Province Hospital of Traditional Chinese Medicine, Zhengzhou Henan 450002 , China;
3. Department of Laboratory Diagnosis, the Second Affiliated Hospital of Dalian Medical University, Dalian Liaoning 116027, China

Published:2013-07-25 Online:2014-03-03
Contact: MENG Xiu-xiang E-mail: xiuxiang_meng@sina.com

摘要/Abstract

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。

关键词: 肺腺癌, 小干扰RNA, Bmi-1基因

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.

Key words: Lung adenocarcinoma, Short interference RNA, Bmi-1 gene

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.

ZHENG Xiang-yu,ZHU Jie,WANG Yi-fang,LIU Chun-qing,LIU Ben,YANG Chun-hui,LIU Dan-dan,MENG Xiu-xiang. Effect of siRNA-mediated silencing Bmi-1 gene expression on the proliferation of lung cancer cell line A549 in vitro and in vivo[J]. China Oncology, 2013, 23(7): 505-511.

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Bmi-1-siRNA对肺腺癌A549细胞体内外增殖能力的影响

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

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