摘要
目的探讨FAM92A1-289对人脑胶质瘤U251细胞增殖、迁移能力的影响及其与半乳糖凝集素1(Galectin-1)的关系。方法将CMV-SP6-TALEN-GFP-289质粒转染至U251细胞中,通过嘌呤霉素筛选得到稳定过表达FAM92A1-289基因的脑胶质瘤U251/289++细胞株。取脑胶质瘤U251/289++细胞作为观察组,未转染质粒的野生型U251细胞作为对照组,采用实时无标记动态细胞分析技术检测两组培养24、48、72、96、120 h的细胞增殖能力(以光密度值表示),采用Transwell小室试验检测两组细胞迁移能力(以穿膜细胞数表示)。构建PCS2-3Flag-Galectin-1质粒,将人脑胶质瘤U251细胞随机分为FAM92A1-289组、Galectin-1组、共转染组及空白对照组,FAM92A1-289组、Galectin-1组均采用Lipofactamine 3000转染法分别转染CMV-SP6-TALEN-GFP-289质粒、PCS2-3Flag-Galectin-1质粒,共转染组同时转染上述两种质粒,空白对照组仅加入Lipofactamine 3000转染试剂。采用免疫共沉淀实验验证FAM92A1-289与Galectin-1是否存在相互作用。结果两组培养24 h光密度值比较差异无统计学意义(P>0.05);观察组培养48、72、96、120 h光密度值均高于对照组(P<0.05或<0.01)。观察组与对照组穿膜细胞数分别为(242.0±11.41)、(65.40±7.92)个,两组比较P<0.01。共转染组抗GFP蛋白相对表达量明显高于FAM92A1-289组、Galectin-1组及空白对照组(P均<0.01)。结论过表达FAM92A1-289可提高人脑胶质瘤U251细胞增殖和迁移能力;FAM92A1-289与Galectin-1之间的相互作用可能为FAM92A1-289调控人脑胶质瘤U251细胞恶性行为的作用机制。
Objective To explore the function of FAM92A1-289 in regulating the proliferation and migration of glioma cells U251 and its relationship with galectin-1. Methods We transfected the plasmid CMV-SP6-TALEN-GFP-289 into 1）251 cells, and used puromycin to get the stable cell line U251/289 ±± with over-expression of FAM92A1-289 gene. We took U251/289 ＋＋ells as the observation group, and U251 cells without transfection as the control group. The real-time cell analysis （RTCA） technology was used to detect the cell proliferation ability of the two groups （ expressed by the optical density value） at 24, 48, 72, 96, and 120 h; Transwell test was used to detect the cell migration ability of the two groups （expressed by the number of cells crossing the trans-well chamber）. We constructed the PCS2-3Flag-Galectin-1 plasmid, and the U251 cells were randomly divided into FAM92A1-289 group, the Galectin-1 group, co-transfection group, and con- trol group. In the FAM92A1-289 group and Galectin-1 group, we used the Lipofactamine 3000 transfection method to trans- fect CMV-SP6-TALEN-GFP-289 and PCS2-3Flag-Galectin-1 plasmids, respectively. The cells in the co-transfection group were transfected with the two plasmids, and in the control group we only added Lipofactamine 3000 transfection reagent. The interaction between FAM92A1-289 and Galectin-1 protein was verified by Co-Immunoprecipitation. Results There was no statistically significant difference between the two groups cells at 24 h （P 〉 0. 05）. The optical density value at 48, 72, 96 and 120 h was significantly higher than that in the control group （P 〈0.05 or P 〈 0. 01 ）. The number of cells crossing the chamber in the observation group and the control group were 242.0 ± 11.41 and 65.40 ± 7.92, respectively （P 〈0.01 ）. The relative expression of GFP in co-transfection group was significantly higher than that in the FAM92A1- 289 group, Galectin-1 group, and control group （ P 〈 0.01 ）. Conclusion The over-expression of FAM92A1-289 can improve the proliferation and migration of U251 cells, and the interaction between FAM92A1-289 and galectin-1 is used to provide evidence for the role of FAM92A1-289 in regulating the malignant behavior of glioma cells.
出处
《山东医药》
CAS
北大核心
2017年第44期9-13,共5页
Shandong Medical Journal
基金
国家自然科学基金应急管理项目(81641028)
湖北省自然科学基金面上项目(2016CFB408)
