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Effect of RNA interference-based silencing of PIN1 gene on proliferation, cell cycle and tumorigenicity of lung cancer A549 cells
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Abstract:
Objective:To study the effect of PIN1 （protein interacting with N1MA1） gene on proliferation, cell cycle and tumorigenicity of lung cancer A549 cells by silencing PIN1 gene using RNA interference technique. Methods: The recombinant plasmid expressing short hairpin RNA (shRNA) targeting PIN1 gene was constructed and named pGPU6-GFP-Neo-PIN1. A549 cells were transfected with pGPU6-GFP-Neo-PIN1 and the negative control plasmid (pGPU6-GFP-Neo) by lipofectamine 2 000. Stable cell lines expressing PIN1 shRNA were obtained after G418 screening. Real-time PCR and Western blotting analysis were performed to determine the expressions of PIN1 at mRNA and protein levels, respectively. Proliferation and cell cycle distribution of A549 cells were detected by MTT assay and flow cytometry assay. Meanwhile, the growth of subcutaneously implanted tumors was observed in nude mice after inoculated with A549 cells with PIN1 stably silenced and the control A549 cells. Results: pGPU6-GFP-Neo-PIN1 plasmid vector was successfully constructed and transfected into A549 cells. PIN1 mRNA expression in A549 cells stably transfected with pGPU6-GFP-Neo-PIN1 decreased by 89.3% compared with that in pGPU6-GFP-Neo-transfected A549 cells, and PIN1 protein was also inhibited by pGPU6-GFP-Neo-PIN1 transfection. Proliferation of PIN1 -silenced A549 cells was significantly suppressed (P<0.01), and their cell cycle was arrested in G1 phase. The tumorigenicity of A549 cells in nude mice was inhibited when PIN1 was silenced (P<0.01). Conclusion: pGPU6-GFP-Neo-PIN1 plasmid stably transfecting into lung cancer A549 cells can effectively silence PIN1 gene expression, inhibit cell proliferation, influence cell cycle and inhibit tumorigenicity of A549 cells.
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