MiR-130b在胶质瘤中的表达及对血管生长的影响

MiR-130b is Down-regulated in Glioma and Inhibits Angiogenesis

目的探讨微小RNA-130b(microRNAs-130b,miR-130b)在胶质瘤细胞中的表达以及调控体外胶质瘤细胞血管生长的作用。方法利用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)法检测miR-130b在胶质瘤细胞株U251、SHG-44和U87及人正常星形胶质细胞株NHA中转录水平的表达。使用miR-130b模拟物(miR-130b mimics)或miR-130b抑制剂(miR-130b inhibitor)瞬时转染体外胶质瘤细胞。提取胶质瘤细胞的条件培养基(conditioned medium,CM),酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)法检测CM中血管内皮生长因子A(vascular endothelial growth factor-A,VEGFA)的表达。CM作用人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)后,Transwell小室实验检测体外HUVECs运动能力。小管形成实验检测HUVECs诱导血管新生的能力。通过生物信息学工具分析miR-130b可能靶基因,荧光素酶报告实验测定荧光素酶活性。凝胶迁移实验(electrophoretic mobility shift assay,EMSA)检测核因子-κB(nuclear factor-κB,NF-κB)活性。Western blot法测定肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和VEGFA蛋白在胶质瘤细胞中的表达水平。结果NHA细胞中miR-130b的表达水平分别为U251、SHG-44和U87细胞的4.29、7.23和10.30倍,差异均有统计学意义(P=0.000)。U87细胞经转染miR-130b模拟物后可明显下调CM中VEGFA表达水平,同时抑制HUVECs体外侵袭和小管形成能力。U251细胞经转染miR-130b抑制剂后可显著上调CM中VEGFA表达水平,并促进HUVECs体外侵袭和小管形成能力。荧光素酶报告实验验证TNF-α是miR-130b的直接作用靶点。与模拟物NC组比较,转染miR-130b模拟物后U87细胞中NF-κB活性、TNF-α和VEGFA蛋白表达均明显下调。与抑制剂NC组比较,转染miR-130b抑制剂后U251细胞中NF-κB活性、TNF-α和VEGFA蛋白表达均显著上调。结论miR-130b在胶质瘤细胞中表达下调并通过靶向调控TNF-α/NF-κB/VEGFA通路抑制体外胶质瘤细胞血管生长。

Objective To investigate the level of miR-130b and its effect in glioma angiogenesis.Methods The relative level of miR-130b in three glioma cell lines(U251,SHG-44 and U87),along with one normal glial cells line NHA was assessed by qRT-PCR.MiR-130b-overexpression U87 cells and miR-130b-knockdown U251 cells were established by transiently transfecttion with miR-130b mimics and miR-130b inhibitor,respectively,and ELISA was used to detect the level of VEGFA in CM.The transwell assay and tubule formation assay were used to evaluate the invasive and angiogenic activity of HUVECs.Bioinformatics software was used to predict the potential target gene of miR-130b,and such predication was validated using luciferase reporter assay.EMSA was performed to detect NF-κB′s DNA binding ability.Western blot was used to assess the levels of TNF-αand VEGFA in glioma cells.Results The expression of miR-130b was markedly decreased in glioma cell lines in comparison with NHA.Transfection of miR-130b inhibitor resulted in an accumulation of VEGFA in U87 cells.Meanwhile,the level of VEGFA was markedly decreased in U251 cells after miR-130b mimics transfection.Over-expression of miR-130b could decrease glioma cells′ability to induce invasion and tube formation of HUVECs.In contrast,the angiogenic ability of HUVECs was promoted significantly by miR-130b knockdown.Luciferase assay confirmed that TNF-αwas a direct target gene of miR-130b.Knockdown of miR-130b in U251 cells significantly promoted,while ectopic expression of miR-130b in U87 cells reduced,NF-κB′s DNA binding ability as well as the level of VEGFA.Conclusion The expression of miR-130b is significantly decreased in glioma cell lines and inhibits glioma angiogenesis by targeting TNF-α/NF-κB/VEGFA pathway.