miR-152通过HLA-G/KIR2DL4通路对dNK分泌LIF和GM-CSF的影响  

The effect of miR-152 on the secretion of LIF and GM-CSF by dNK through the HLA-G/KIR2DL4 pathway

作　　者：杨洋 陈宥艺[1] 宋姗姗 张欣文[3] YANG Yang;CHEN You-yi;SONG Shan-shan;ZHANG Xin-wen(Reproductive Medicine Center,Xi'an People's Hospital(Xi’an Fourth Hospital),Xi'an,Shaanxi 710004;Xi'an Medical University,Xi'an,Shaanxi 710021;Department of Obstetrics,Xi'an People's Hospital(Xi'an Fourth Hospital),Xi'an,Shaanxi 710004,China)

机构地区：[1]西安市人民医院(西安市第四医院)生殖医学中心,陕西西安710004 [2]西安医学院,陕西西安710021 [3]西安市人民医院(西安市第四医院)妇产科,陕西西安710004

出　　处：《热带医学杂志》2022年第5期625-629,共5页Journal of Tropical Medicine

基　　金：国家自然科学基金(81901510);陕西省自然科学基础研究计划(2020JM-615);西安市科技计划项目[20YXYJ0005(6)]

摘　　要：目的研究微小RNA-152(miR-152)通过其靶基因人类白细胞抗原G(HLA-G)与蜕膜NK细胞(dNK)表面杀伤细胞免疫球蛋白样受体(KIR)KIR2DL4之间通路,对dNK分泌白血病抑制因子(LIF)及粒细胞-巨噬细胞集落刺激因子(GM-CSF)的影响。方法收集2020年9月-2020年10月在西安市人民医院行人工流产患者的正常早孕蜕膜组织5例,分选、纯化dNK。在体外培养的HTR8细胞中过表达和抑制miR-152,分为对照组(未转染)、过表达组(转染miR-152 mimics)与抑制组(转染miR-152 inhibitor)。实时定量PCR(qRT-PCR)检测转染后miR-152水平。构建dNK与过表达或抑制miR-152后的HTR8共培养体系,并在共培养过程中特异性封闭dNK KIR2DL4,分组为:NC组[HTR-8+dNK+白细胞介素-15(IL-15)共培养]、A组(过表达miR-152的HTR-8+dNK+IL-15)、B组(抑制miR-152的HTR-8+dNK+IL-15)、C组[过表达miR-152的HTR-8+dNK+KIR2DL4抑制物(anti-KIR2DL4)+IL-15]、D组[过表达miR-152的HTR-8+dNK+IgG1(anti-KIR2DL4对照物)+IL-15]、E组(转染miR-152 mimics NC的HTR-8+dNK+antiKIR2DL4+IL-15)。酶联免疫吸附测定(ELISA)检测共培养24 h后上清中LIF、GM-CSF的表达。结果转染后6 h于荧光显微镜下观察转染效率(羧基荧光素酶标记对照),可见转染效率超过90%。与对照组(1.27±0.29)相比,过表达组细胞中miR-152表达水平升高(2.63±0.44),抑制组细胞中miR-152表达降低(0.34±0.12),差异均有统计学意义(F=130.47、130.47,P<0.05)。与NC组相比,C组共培养上清中LIF、GM-CSF的浓度最低;A、D、E组在过表达miR-152的前提条件下,共培养上清中LIF、GM-CSF的浓度均分别低于NC组;B组共培养上清中LIF、GM-CSF的浓度高于NC组,差异均有统计学意义(P<0.05)。结论miR-152能够通过HLA-G/KIR2DL4通路影响dNK分泌LIF、GM-CSF。Objective To study the effect of microRNA-152(miR-152)on the secretion of leukemia inhibitory factor(LIF)and granulocyte-macrophage colony-stimulating factor(GM-CSF)from decidual natural killer(dNK)through the pathway between its target gene human leukocyte antigen-G(HLA-G)and the killer Ig-like receptors(KIR)KIR2DL4 on the surface of decidual NK cells.MethodsFive patients undergoing induced abortion in Xi’an People’s Hospital from September 2020to October 2020 were collected.Human normal early pregnancy decidua tissues were collected to sort and purify dNK.MiR-152 was overexpressed and inhibited in HTR8 cells cultured in vitro,and they were divided into control group(no transfected),overexpression group(transfected with miR-152 mimics)and inhibition group(transfected with miR-152inhibitor).Real-time quantitative PCR(qRT-PCR)was used to detect the level of miR-152 after transfection.The coculture system of dNK cells and HTR8 after overexpression or inhibition of miR-152 was constructed,and KIR2DL4 on the surface of dNK was specifically blocked during the co culture process;they were divided into NC group[HTR-8+dNK+interleukin-15(IL-15)],group A(HTR-8 overexpressing miR-152+dNK+IL-15),group B(HTR-8 inhibiting miR-152+dNK+IL-15),group C[HTR-8 overexpressing miR-152+dNK+KIR2DL4 inhibitor(anti-KIR2DL4)+IL-15],group D[HTR-8 overexpressing miR-152+dNK+IgG1(anti-KIR2DL4 control)+IL-15],group E(HTR-8 transfected with miR-152mimics NC+dNK+anti-KIR2DL4+IL-15).Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of LIF and GM-CSF in the supernatant after 24 hours of co-culture;transwell experiment was used to detect the effect of coculture supernatant on the infiltration capacity of HTR8.ResultsThe transfection efficiency(carboxy luciferase labeling control)was observed under a fluorescence microscope 6 h after transfection,and the transfection efficiency was more than90%.Compared with the control group(1.27±0.29),the expression level of miR-152 in the overexpression group cells increased(2.63±0.4

关 键 词：微小RNA 蜕膜NK细胞 白血病抑制因子 粒细胞-巨噬细胞集落刺激因子 

分 类 号：R714.244[医药卫生—妇产科学]