miR-152通过影响dNK分泌GM-CSF抑制人脐静脉内皮细胞功能

The miR-152 inhibits the function of human umbilical vascular endothelial cell through granulocyte-macrophage colony stimulating factor secreted by decidual natural killer

目的研究微小RNA(miR-152)通过蜕膜NK细胞(decidual natural killer,dNK)分泌粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)对人脐静脉内皮细胞(human umbilical vascular endothelial cells,HUVECs)功能的影响。方法为研究miR-152对人类白细胞抗原G(human leukocyte antigen-G,HLA-G)的调控作用,将人绒毛膜滋养细胞(HTR8)分为过表达组(转染miR-152 mimics)、抑制组(转染miR-152 inhibitor)、对照组(空白转染);实时定量PCR和蛋白免疫印迹检测转染后miR-152及HLA-G在mRNA和蛋白水平的表达。为研究miR-152影响dNK分泌功能的可能方式,将从人正常早孕蜕膜中提取的dNK与转染miR-152 mimics及inhibitor的HTR8分组共培养,并在共培养过程中加入杀伤细胞免疫球蛋白样受体2DL4(killer Ig-like receptors,KIR2DL4)封闭剂以阻断HLA-G/KIR2DL4通路,并以KIR2DL4封闭剂对照物免疫球蛋白G1(immune gamma globulins,IgG1)作为封闭后对照,具体分组:空白共培养组、miR-152过表达共培养组、miR-152过表达对照组、miR-152抑制共培养组、通路封闭共培养组、通路封闭对照组。ELISA检测共培养24 h后上清中GM-CSF的表达;管样形成实验检测共培养上清对人脐静脉内皮细胞管样形成过程中总分支长度及管腔数的影响。结果与对照组相比,过表达组miR-152在HTR8细胞中表达显著增高(P<0.01),抑制组中miR-152在表达显著降低(P<0.01)。与对照组相比,过表达组HLA-G在mRNA及蛋白水平均显著降低(P<0.01),抑制组HLA-G在mRNA及蛋白水平均显著升高(P<0.01)。与空白共培养组相比,miR-152抑制共培养组上清中GM-CSF的浓度明显增高(P<0.01)。与空白共培养组比较,通路封闭共培养组上清干预后HUVEC总分支长度及管腔数均明显降低(P<0.01),miR-152抑制共培养组上清干预后HUVEC总分支长度及管腔数均明显增高(P<0.01)。结论过表达miR-152能够影响dNK细胞分泌GM-CSF,从而抑制HUVEC成管能力。

Objective To explore the effect of microRNA 152(miR-152)on the function of human umbilical vein endothelial cells(HUVECs)through regulating granulocyte-macrophage colony stimulating factor(GM-CSF)secreted by the decidual natural killer cells(dNK).Methods Human chorionic trophoblasts(HTR8)were divided into three groups:overexpression group(transfected with miR-152 mimics),inhibitor group(transfected with miR-152 inhibitor)and control group(blank transfection).The expression of miR-152 and HLA-G at mRNA and protein levels was detected using real-time quantitative PCR and Western blotting in the three groups to clarify the effect of miR-152 on human leukocyte antigen-G(HLA-G).The dNK was extracted from normal human early pregnancy decidua and co-cultured with HTR8 transfected with miR-152 mimics and inhibitor.The killer Ig-like receptors 2DL4(KIR2DL4)blocker was also added into the co-culture to block the HLA-G/KIR2DL4 pathway.Meanwhile the KIR2DL4 blocker control substance immunoglobulin G1(immune gamma globulins,IgG1)was used as the controls.Ultimately,they were divided into six groups:blank co-culture group,miR-152 mimics co-culture group,miR-152 mimics control group,miR-152 inhibitor co-culture group,pathway blocker co-culture group,and pathway blocker control group.ELISA was used to analyze the level of GM-CSF in the supernatant after co-culture for 24 h,and the tube formation experiment was used to explore the total branch length and the number of lumens in the process of HUVEC tube formation.Results Compared with control group,the expression of miR-152 in overexpression group was significantly increased(P<0.01),while the expression of miR-152 in inhibitor group was significantly decreased(P<0.01).Compared with control group,the expression of HLA-G at mRNA and protein levels was significantly decreased in overexpression group(P<0.01),while the expression of HLA-G at mRNA and protein levels was significantly increased in inhibitor group(P<0.01).The concentration of GM-CSF in the supernatant in miR-152 inhibition co-culture group was significantly higher than that in blank co-culture group(P<0.01).Compared with blank co-culture group,the total branch length and the number of lumens of HUVECs were significantly reduced after the supernatant intervention in miR-152 overexpression and pathway blocker co-culture group(P<0.01),while the total branch length and the number of lumens of HUVECs were significantly increased in miR-152 inhibitor and pathway blocker co-culture group(P<0.01).Conclusion Overexpression of miR-152 can affect the secretion of GM-CSF by dNK cells,thereby inhibiting the tube formation ability of HUVECs.