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[Establishment of a lung cancer A549 cell line stable over-expressing integrin linked kinase protein and its
Hai-Yan Hu1, Quan Chen, Yan Chen
1Department of Immunity, College of Basic Medicine, Chongqing Medical University, Chongqing 400016, China. huhaiyan22@163.com
Aim:
To establish a lung cancer A549 cell line stable over-expressing human integrin linked kinase (ILK) and study the effect of over-expression of ILK on biological activity of A549 cells.
Methods:
Human ILK gene was amplified by RT-PCR, then cloned into pEGFP-C1 vector to construct pEGFP-ILK. After confirmed by restriction analysis and sequencing, the recombinant plasmid was transfected into A549 cells mediated with liposome, then G418-resistant clones of A549 cells (A549/pEGFP-ILK) as experimental group were obtained, and paralleled with the vector control (A549/pEGFP-C1) and A549 cell control. The expression and localization of EGFP-ILK fusion protein in A549 cells was observed by fluorescence microscopy. RT-PCR and Western blot were performed to detect the level of ILK mRNA and ILK protein of each group cells respectively. The cell proliferation was tested by methylthiazolyl tetrazolium (MTT) assay, and the cell apoptosis was measured by flow cytometry, and the morphologic changes of cells were observed by HE staining.
Results:
Both restriction analysis and sequencing proved that the pEGFP-ILK plasmid was constructed correctly. The distribution of fluorescence of stable transfected A549 cells indicated that the product of ILK gene was mainly located in cytoplasm. Compared with A549/pEGFP-C1 group and A549 group, the level of ILK mRNA and ILK protein of A549/pEGFP-ILK cells were significantly increased, which over-expression ratio was 218.18% and 245.45% respectively (P<0.05). The proliferation ability of the A549 cells over-expressing ILK was increased significantly (P<0.05). However, the apoptosis of A549/pEGFP-ILK cell was inhibited significantly by over-expression of ILK (P<0.05). After HE staining, the increased mitosis were observed only in A549/pEGFP-ILK group cells.
Conclusion:
The lung cancer cell line stable over-expressing ILK protein was constructed successfully, and ILK over-expression could promote cell proliferation, inhibit cell apoptosis and increase mitosis.
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