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目的:研究桥接整合因子1(bridging intergrator-1,Bin1)基因过表达对非小细胞肺癌细胞株A549细胞迁移和侵袭能力的影响,并初步探讨其作用机制。方法:通过基因转染技术,利用阳离子脂质体将含有人全长Bin1基因序列的真核表达质粒CMV-MCS-GFP-SV40-Neomycin-Bin1转染到A549细胞株,分别设置空白对照组及空质粒转染组,利用RT-PCR和Western blotting分别检测各处理组细胞中Bin1基因和蛋白表达水平。通过细胞划痕实验、Transwell侵袭实验分别检测Bin1过表达对A549细胞迁移、侵袭能力的影响;Western blotting实验检测Bin1过表达对A549细胞内NF-κB磷酸化水平和迁移相关蛋白E-钙黏着蛋白、N-钙黏着蛋白、MMP-9表达水平的影响。结果:与空白对照组和空质粒转染组相比,Bin1转染组A549细胞中Bin1基因和蛋白表达水平均明显升高(P<0.05);Bin1转染组细胞迁移、侵袭能力均较空白质粒组和空白对照组明显下降[穿膜细胞数:(50.50±3.15)vs(124.00±4.25),(130.00±4.37)个;均P<0.05];与空白转染组和空白对照组相比,Bin1转染组细胞内NF-κB表达水平明显上调(P<0.05)而p-NF-κB表达明显下调(P<0.05),N-钙黏着蛋白、MPP-9明显下调(P<0.05),E-钙黏着蛋白明显上调(P<0.05)。结论:Bin1过表达可以抑制A549细胞的迁移及侵袭能力,其机制可能与NF-κB途径的失活及细胞迁移侵袭相关蛋白表达变化有关。
OBJECTIVE: To study the effect of bridging intergrator-1 (Bin1) gene overexpression on the migration and invasion of non-small cell lung cancer cell line A549 and to explore its possible mechanism. Methods: The eukaryotic expression plasmid CMV-MCS-GFP-SV40-Neomycin-Bin1 containing the full-length Bin1 gene was transfected into A549 cell line by cationic liposome transfection. The blank control group and control group Empty plasmid transfected group, the expression of Bin1 gene and protein in each treatment group were detected by RT-PCR and Western blotting respectively. Transwell invasion assay was used to detect the effect of Bin1 overexpression on the migration and invasion ability of A549 cells. Western blotting was used to detect the effect of Bin1 overexpression on NF-κB phosphorylation and migration-associated protein E-cadherin , N-cadherin, MMP-9 expression levels. Results: Bin1 mRNA and protein expression were significantly increased in Bin1-transfected A549 cells compared with those in blank control group and blank plasmid transfected group (P <0.05). The migration and invasion ability of Bin1-transfected A549 cells were more than blank The number of transmembrane cells was significantly lower in the plasmid group and the blank control group (50.50 ± 3.15 vs 124.00 ± 4.25, 130.00 ± 4.37, respectively; all P <0.05). Compared with the blank transfected group and the blank control group, The expression of NF-κB in Bin1 transfection group was significantly increased (P <0.05), and the expression of p-NF-κB was significantly decreased (P <0.05). The N-cadherin and MPP- , E-cadherin was significantly increased (P <0.05). CONCLUSION: Bin1 overexpression can inhibit the migration and invasion of A549 cells. The mechanism may be related to the inactivation of NF-κB pathway and the changes of protein expression related to cell migration and invasion.