Establishment Of A Lung Cancer A549Cell Line Stable Over-Expressing Integrin-Linked Kinase Protein And Its Biological Activity With Mechanism

Objective1. To establish a lung cancer A549cell line stable over-expressing human Integrin-Linked Kinase (ILK).2. To study the effect of over-expression of ILK on biological activity of A549cells and its mechanism.Methods1. Human ILK gene was amplified by RT-PCR. After restriction endonuclease digestion with XhoTå'ŒSalT, ILK gene was inserted pEGFP-C1vector to construct pEGFP-ILK.2. After confirmed by restriction analysis and sequencing, the pEGFP-ILK eukaryotic expression plasmid was transfected into A549cells mediated with liposome, then G418-resistant clones of A549cells (A549/pEGFP-ILK) as experimental group were obtained, and paralleled with the vector control (A549/pEGFP-C1) and A549cell control.3. The expression and localization of EGFP-ILK fusion protein in A549cells was observed by fluorescence microscopy. RT-PCR were performed to detect the level of ILK mRNA and the expression level of ILK, p-GSK3β and β-catenin of each group cells were assessed by Western-blot. 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.Results1. Both restriction analysis and sequencing proved that the pEGFP-ILK plasmid was constructed correctly.2. The distributing of fluorescence of stable transfected A549cells indicated that the product of ILK gene was mainly located in cytoplasm. Compared with A549/pEGFP-C1group and A549group, the level of ILK mRNA and ILK protein of A549/pEGFP-ILK cells were significantly increased, which over-expression ratio was218.18%and245.45%respectively (P<0.05). The expression level of p-GSK3β and P-catenin protein were increased47.62%and68.69%respectively.3. The proliferation ability of the A549cells 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.Conclusions1. 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.2. The possible mechanism that ILK regulated biological activity of lung cancer was by mediating the expression of downstream signal transduction molecules GSK3and P-catenin. The lung cancer cell model stable over-expressing Integrin-linked kinase protein can be used to research into biological activity and mechanism of ILK.