miR-455-5p靶向RECK基因对A549细胞迁移及微血管形成的影响

Effect of miR-455-5p targeting RECK gene on A549 cell migration and microvascular angiogenesis

目的探讨miR-455-5p靶向RECK基因对人非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞A549迁移及微血管形成的影响。方法利用UALCAN网站对TCGA数据库中肺癌miR-455-5p和RECK基因表达数据进行分析,miRanda在线预测miR-455-5p与RECK基因的靶向关系,并通过双荧光素酶报告基因试验进行靶点验证;A549细胞中分别转染miR-455-5p模拟物、抑制剂及阴性对照后,qRT-PCR检测mi R-455-5p及RECK基因mRNA的表达,Western blot法检测RECK、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)和MMP9蛋白的表达;TranswellTM小室试验检测A549细胞的迁移能力;ELISA法检测A549细胞培养上清液中血管内皮生长因子(vascular endothelial growth factor,VEGF)-A的表达;用A549细胞培养上清液作用人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC),体外微管形成试验检测微血管形成能力。结果肺癌组织中miR-455-5p表达显著高于正常组织,而RECK基因的表达显著低于正常组织,miR-455-5p在RECK基因的3′-UTR区有结合域,RECK为miR-455-5p直接调控靶基因。miR-455-5p下调靶基因RECK的表达,而上调MMP2、MMP9蛋白表达;miR-455-5p可明显促进A549细胞迁移及分泌VEGF-A,并增强其诱导HUVEC分化成管的能力。结论mi R-455-5p通过靶向RECK上调MMPs和VEGF-A,来促进NSCLC细胞A549的迁移及微血管形成。

Objective To investigate the effect of miR-455-5p targeting RECK gene on migration and microvascular angiogenesis of non-small-cell lung carcinoma(NSCLC)A549 cells.Methods The data on expressions of mi R-455-5p and RECK genes in lung cancer in TCGA database were analyzed by UALCAN online,and the targeting relationship between miR-455-5p and RECK genes was predicted by miRanda.The target was validated by double luciferase reporter gene assay.A549 cells were transfected with miR-455-5p mimic,inhibitor and negative control respectively,and determined for the relative expressions of miR-455-5p and RECK mRNAs by qRT-PCR,and for the expressions of RECK,MMP2 and MMP9 by Western blot.The migration ability of A549 cells was determined by TranswellTMassay.The expression of vascular endothelial growth factor(VEGF)-A in culture supernatant of A549 cells was determined by ELISA.Human umbilical vein endothelial cells(HUVECs)were treated with the culture supernatant of A549 cells and determined for microvascular angiogenesis ability by in vitro microtube formation assay.Results The expression level of miR-455-5p was significantly higher,while that of RECK was significantly lower in lung cancer tissue than in normal tissues.The miR-455-5p had a binding domain in the 3′-UTR region of RECK gene which was the direct target.The miR-455-5p down-regulated the expression of target gene RECK while up-regulated those of MMP2 and MMP9,promoted the migration of A549 cells and the secretion of VEGF-A significantly,and enhanced the ability of inducing HUVEC to differentiate into microtubules.Conclusion The miR-455-5p promoted the A549 cell migration and microvascular angiogenesis by up-regulating the expressions of MMPs and VEGF-A via directly targeting RECK.