Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Dec;14(12):903-7.
doi: 10.3779/j.issn.1009-3419.2011.12.01.

[Effect of shRNA Survivin-mediated inhibition of proliferation in A549 cells]

[Article in Chinese]
Lihong Wu  1 , Jinhe Wang, Qian Wang, Dongdong Jia, Jianqin Liang
Affiliations

[Effect of shRNA Survivin-mediated inhibition of proliferation in A549 cells]

[Article in Chinese]
Lihong Wu et al. Zhongguo Fei Ai Za Zhi. 2011 Dec.

Abstract
in English, Chinese

Background and objective: Survivin is a member of the inhibitor of apoptosis family of proteins. The Survivin protein is highly expressed in most human tumors, but it is completely absent in terminally differentiated cells. Consequently, Survivin is an ideal target for cancer therapy because cancer cells are targeted and normal cells are left alone. The aim of this study is to construct a lentivirus-shRNA vector, and to disrupt the expression of Survivin in A549 cells. The effect of sh-RNA Survivin on A549 cells was analyzed.

Methods: Target DNA sequences of Survivin shRNA were designed to obtain recombinant plasmids. After the plasmids were transfected into 293T cells, the virus was collected. Hela cells were used to detect the virus titer. Survivin mRNA and protein expression in the infected A549 cells were detected via reverse transcription polymerase chain reaction and Western blot analysis. The proliferation of A549 cells were detected via 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide and flow cytometry assays.

Results: The recombinants were successfully constructed, and Survivin expression was inhibited. The cells were blocked at the G2/M phase.

Conclusions: Recombinant lentivirus with shRNA targeting Survivin was successfully constructed. The lentivirus can down-regulate Survivin expression in A549 cells as well as inhibit proliferation, and is hence a potential gene therapy for lung cancer.

背景与目的: Survivin是凋亡抑制蛋白(inhibitor of apoptosis protein, IAP)家族的成员, 在多种肿瘤组织中高度表达而在终末分化细胞中极少表达, 因此可以作为癌症治疗的理想靶点。本研究旨在通过构建Survivin基因shRNA的慢病毒质粒并干扰肺癌细胞A549中Survivin的表达, 分析其对细胞增殖的影响。

方法: 设计Survivin干扰靶序列, 构建重组质粒; 将pLL3.7-Survivin转染293T细胞后利用Hela细胞检测病毒的滴度并感染A549细胞, 应用RT-PCR和Western blot检测干扰效果; MTT与流式细胞术分析其对细胞增殖的影响。

结果: 本研究成功构建了重组质粒; 重组质粒可抑制A549细胞中Survivin基因的表达; 细胞受阻于G2/M期。

结论: 本研究构建的重组质粒可抑制Survivin基因的表达并影响细胞的增殖, 其为研究RNAi介导的肺癌基因治疗打下基础。

PubMed Disclaimer

Figures

1
1
重组质粒双酶切结果 Double enzyme cutting result of recombinant plasmids.M:Marker; 1:pll3.7;2:pll3.7-Control; 3:pll3.7-Survivin1;4:pll3.7-Survivin2.
2
2
转染效率及病毒滴度检测结果。A:荧光检测转染的293T细胞(左)和明场观察(右); B:荧光检测感染Hela细胞(左)和明场观察(右)。 Results of transfection efficiency and virus titer.A:293T cell shot by fluorescent light (left) and light (right); B:Hela cell shot by fluorescent light (left) and light (right).
3
3
RT-PCR及Western blot检测结果。A:RT-PCR检测Survivin mRNA的表达, 柱状图为相对内参GAPDH的灰度值; 与Control组相比, *P < 0.05。B:Western blot检测Survivin表达量。与Control相比, *P < 0.05。 Results of RT-PCR (A) and Western blot (B).A:The result of Survivin mRNA RT-PCR, anlysis of expression compare with GAPDH; *P < 0.05, vs Control.B:The result of Survivin expression by Western blot.1:PBS; 2:pLL3.7-Control; 3:pLL3.7-Survivin1;4:pLL3.7-Survivin2.
4
4
流式细胞术检测细胞周期。 Results of flow cytometry.A:PBS; B:pLL3.7-Control; C:pLL3.7-Suivivin2;D:results of cell cycle.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Sah NK, Khan Z, Khan GJ, et al. Structural, functional and therapeutic biology of Surviving. Cancer Lett. 2006;244(2):164–171. doi: 10.1016/j.canlet.2006.03.007. - DOI - PubMed
    1. Altieri DC. Validating Survivin as a cancer therapeutic target. Nat Rev Cancer. 2003;3(1):46–54. doi: 10.1038/nrc968. - DOI - PubMed
    1. Harborth J, Elbashir SM, Vandenburgh K, et al. Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev. 2003;13(2):83–105. doi: 10.1089/108729003321629638. - DOI - PubMed
    1. Shin S, Sung BJ, Cho YS, et al. An anti-apoptotic proteinhuman Survivin is a direct inhibitor of caspase-3 and -7. Biochemistry. 2001;40(4):1117–1123. doi: 10.1021/bi001603q. - DOI - PubMed
    1. Suzuki A, Ito T, Kawano H, et al. Survivin initiates pro-caspase3/p21 complex formation as a result of interaction with Cdk4 to resist Fas-medited cell death. Oncogene. 2000;19(10):1346–1353. doi: 10.1038/sj.onc.1203429. - DOI - PubMed

MeSH terms