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目的:应用RNA干扰技术沉默肺癌A549细胞中PIN1(protein interacting with N1MA1)基因的表达,探讨其对A549细胞增殖、细胞周期和裸鼠成瘤能力的影响。方法:构建靶向PIN1基因的shRNA真核表达质粒pGPU6-GFP-Neo-PIN1和无义对照质粒pGPU6-GFP-Neo,以脂质体法转染A549细胞,G418筛选稳定沉默PIN1基因的细胞株。Real-time PCR和Western blotting验证PIN1基因在mRNA和蛋白水平的表达,MTT法和流式细胞术检测A549细胞增殖和细胞周期分布。将稳定沉默PIN1的A549细胞与对照细胞皮下接种裸鼠,观察接种后肿瘤生长情况。结果:成功构建了pGPU6-GFP-Neo-PIN1载体,转染A549细胞并筛选获得稳定克隆。稳定转染pGPU6-GFP-Neo-PIN1的A549细胞中PIN1mRNA表达量较pGPU6-GFP-Neo转染组下降了89.3%;蛋白表达同时也显著抑制。PIN1基因沉默组的A549细胞增殖速率明显下降(P<0.01),细胞出现G1期阻滞。小鼠体内实验显示,PIN1沉默的A549细胞在裸鼠体内成瘤能力降低(P<0.01)。结论:pGPU6-GFP-Neo-PIN1质粒稳定转染肺癌A549细胞能有效沉默PIN1基因的表达,从而抑制A549细胞的增殖、影响细胞周期和抑制成瘤能力。
Objective: To study the effect of RNA interference on the expression of PIN1 (protein interacting with N1MA1) gene in lung cancer A549 cells and to explore its effect on the proliferation, cell cycle and tumorigenicity of A549 cells. Methods: The eukaryotic expression plasmid pGPU6-GFP-Neo-PIN1 targeting PIN1 gene and the pGPU6-GFP-Neo non-sense control plasmid were constructed and transfected into A549 cells by lipofectamine. The cell lines stably silenced by PIN1 were screened by G418 . The expression of PIN1 gene at mRNA and protein level was verified by Real-time PCR and Western blotting. The proliferation and cell cycle distribution of A549 cells were detected by MTT assay and flow cytometry. The A549 cells stably silenced with PIN1 and the control cells were subcutaneously inoculated with nude mice to observe the tumor growth after inoculation. Results: The pGPU6-GFP-Neo-PIN1 vector was successfully constructed, transfected into A549 cells and screened to obtain stable clones. Compared with pGPU6-GFP-Neo transfection group, the expression of PIN1 mRNA in A549 cells stably transfected with pGPU6-GFP-Neo-PIN1 was decreased by 89.3%, and protein expression was also significantly inhibited. The proliferation rate of A549 cells in the PIN1 gene silencing group was significantly decreased (P <0.01), and G1 phase arrest occurred in the cells. Mouse in vivo experiments showed that PIN1-silenced A549 cells in vivo nude mice ability to reduce tumor formation (P <0.01). CONCLUSION: The plasmid pGPU6-GFP-Neo-PIN1 stably transfected A549 cells can effectively silence the expression of PIN1 gene, thereby inhibiting the proliferation of A549 cells, affecting cell cycle and inhibiting tumorigenicity.