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. 2012 Dec;15(12):694-700.
doi: 10.3779/j.issn.1009-3419.2012.12.03.

[Construction of a CD147 lentiviral expression vector and establishment of its stably transfected A549 cell line]

[Article in Chinese]
Shaoxing Yang  1 , Chuanhao Tang, Sihan Wang, Santai Song, Xiaoqing Liu
Affiliations

[Construction of a CD147 lentiviral expression vector and establishment of its stably transfected A549 cell line]

[Article in Chinese]
Shaoxing Yang et al. Zhongguo Fei Ai Za Zhi. 2012 Dec.

Abstract
in English, Chinese

Background and objective: CD147, a type of transmembrane glycoprotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells.

Methods: Full-length CD147 gene was amplified by real-time polymerase chain reaction (RT-PCR), inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable, overexpressed cell line named A549-CD147. The mRNA expression of MMP-9 was examined by RT-PCR. The proliferation and invasive ability of the human lung cancer cells before and after transfection were examined by the CCK-8 and Transwell methods.

Results: A CD147 lentiviral expression vector (pEGFP-CD147) was successfully constructed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blot analyses revealed increased mRNA and protein expression of CD147 gene in cells transfected with pEGFP-CD147 compared with the control groups. Therefore, the A549-CD147 cell line was successfully established through the experiment. The mRNA expression of MMP-9 also significantly increased after the upregulation of CD147 expression. Meanwhile, CCK-8 and Transwell assays indicated that the proliferation and invasive ability significantly increased in the A549-CD147 cells.

Conclusions: A lentiviral CD147 expression vector and its A549 cell line (A549-CD14) were successfully constructed. CD147 overexpression upregulated the protein expression of MMP-9, and strengthened the proliferation and invasive ability of human lung adenocarcinoma cells.

背景与目的: CD147是一类位于肿瘤细胞膜表面的跨膜糖蛋白，可促进肿瘤的浸润和转移。本研究拟构建CD147慢病毒表达载体，建立稳定过表达CD147的人肺腺癌A549细胞系，观察过表达CD147后对MMP-9及细胞增殖、侵袭能力的影响。

方法: RT-PCR扩增CD147基因全长序列，将序列插入pEGFP载体，构建pEGFP-CD147慢病毒表达载体，随后转入293FT细胞中进行慢病毒包装，用获得的慢病毒毒液感染人肺腺癌细胞系A549，建立稳定过表达CD147的A549细胞系。Real-time PCR检测MMP-9的变化情况，CCK-8及Transwell法检测人肺腺癌细胞增殖、侵袭能力的变化。

结果: 经限制性内切酶鉴定及测序分析，成功构建了pEGFP-CD147慢病毒表达载体质粒。Real-time PCR和Western blot检测显示，与对照组相比，转染pEGFP-CD147慢病毒表达载体组的细胞，CD147的表达在mRNA和蛋白两个水平均增高，成功建立了A549-CD147细胞系。上调CD147的表达后，MMP-9的mRNA表达水平明显升高。同时，A549-CD147细胞增殖和侵袭能力明显增加（P < 0.05）。

结论: 成功构建CD147慢病毒表达载体和A549-CD147细胞系，过表达CD147可上调MMP-9的表达，增强人肺腺癌细胞的增殖和侵袭能力。

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Figures

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1
CD147慢病毒表达载体的构建及鉴定 Construction and identification of CD147 lentiviral expression vector. A: Human CD147 whole genome obtained; 1: Human CD147 gene. B: Restriction map analysis of CD147 lentiviral expression vector; 1: pEGFP vector and CD147 gene; C: Identification of CD147 lentiviral expression vector by RT-PCR; 1: CD147 gene.
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2
pEGFP-CD147慢病毒的包装和感染人肺腺癌A549细胞系 pEGFP-CD147 lentiviral packaging and infection of human lung adenocarcinoma cell line A549. A: pEGFP-CD147 lentiviral expression vector was transfected in 293FT cells; B: pEGFP-CD147 lentiviral venom infected A549 cells; C: Detection of CD147 mRNA expression levels in the respective cells by RT-PCR; D: Detection of CD147 mRNA expression levels in the respective cells by real-time PCR.
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A549-CD147细胞系的建立 Established the A549-CD147 cell line. A: The A549 cells transfected with p-EGFP lentiviral expression vector and p-EGFP empty vector were sorted by flow cytometry respectively; B: The A549-pEGFP cells and A549-CD147 cells after sorted by flow cytometry; C: Detection of CD147 mRNA expression levels in the respective cells by real-time PCR; D: Detection of CD147 in protein level by Western blot in cells.
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过表达CD147对MMP-9及人肺癌细胞增殖、侵袭能力的影响 Effects on MMP-9, proliferation and invasive ability of the human lung adenocarcinoma cells after overexpression CD147. A: The relative expression rate of MMP-9 mRNA was significantly enhanced in A549-CD147 cells than that in A549 and A549-pEGFP cells; B: The growth curves indicated that the growth rates had not significant difference among A549-CD147, A549-pEGFP and A549 cells in the first day after transfection, but in the second day (P < 0.05) and the third day (P < 0.01), the growth rate was significantly higher in A549-CD147 cells than that in A549-pEGFP and A549 cells; C: Representative microscope field of filters under the Matrigel from A549-CD147, A549-pEGFP and A549 cells respectively (×100); D: The Histogram showed that the number of invasive cell was significantly more in A549-CD147 cells than that in A549-pEGFP and A549 cells (P < 0.01).
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