Inhibition of HLA-G Expression Via RNAi Abolishes Resistance of Extravillous Trophoblast Cell Line TEV-1 to NK Lysis
Inhibition of HLA-G Expression Via RNAi Abolishes Resistance of Extravillous Trophoblast Cell Line TEV-1 to NK Lysis
复制标题
通过 RNAi 抑制 HLA-G 表达消除绒毛外滋养层细胞系 TEV-1 对 NK 裂解的抗性
DOI：
10.1016/j.placenta.2010.03.008
复制
发表时间：
2010-06-01
期刊：
影响因子：
3.8
通讯作者：
Zou, P.
中科院分区：
文献类型：
--
作者：
Chen, L. J.;Han, Z. Q.;Zou, P.
子宫螺旋动脉的重塑发生在妊娠的前三个月，涉及蜕膜和绒毛外滋养层细胞中母体自然杀伤（NK）细胞的扩增和活化。侵袭性滋养层细胞在其侵入子宫组织期间遇到母体NK细胞，从而引起对NK裂解的易感性的问题。体内外研究表明，侵袭性绒毛外滋养细胞表达HLA-G可能为NK细胞提供了免疫保护，但HLA-G敲低对滋养细胞/NK细胞相互作用的影响尚缺乏直接证据。本研究旨在探讨HLA-G下调对绒毛外滋养细胞的影响。构建靶向HLA-G的短发夹状RNA（shRNA）载体，转染人早孕绒毛外滋养层细胞系TEV-1。Western blotting和RT-PCR检测结果显示，转染HLA-G shRNA的细胞中HLA-G的表达明显降低。HLA-G表达也通过共聚焦成像可视化。用流式细胞仪分析TEV-1细胞的HLA表型和NK细胞抑制性受体的表达。HLA-G敲低和非敲低细胞之间的比较显示在不改变HLA-C和HLA-E的情况下HLA表达谱的显著差异。原代NK细胞和NK-92细胞系均通过标准4-h Cr-51释放测定显示出针对HLA-G敲低细胞的强效细胞毒性。NK细胞中ILT2、ILT4和KIR2DL4的表达在共培养4 h后无变化，而KIR2DL4的表达在48 h后增加。我们的结论是，HLA-G有助于滋养层/NK细胞的相互作用，作为一个关键的调节NK细胞溶解在这个人绒毛外滋养层细胞模型。此外，TEV-1细胞与绒毛外滋养层细胞具有共同的HLA表型特征，因此可用作良好的细胞模型。滋养层细胞中的HLA-C表达与HLA-G翻译无关，单独的HLA-C就足以增强HLA-E表面表达。此外，RNA干扰技术可作为研究HLA-G功能的一种可行而有效的方法。(C)2010爱思唯尔有限公司版权所有。
Remodelling of uterine spiral arteries occurs in the first trimester of pregnancy and involves an expanded and activated population of maternal natural killer (NK) cells in the decidua and extravillous trophoblast cells. Invasive trophoblasts encounter maternal NK cells during their invasion into the uterine tissue, posing the problem of susceptibility to NK lysis. Studies in vitro and in vivo suggested that the expression of HLA-G by invasive extravillous trophoblasts might provide invulnerability to NK cells, while there is still lack of direct evidence of HLA-G knockdown effect on trophoblast/NK interaction. A study was conducted to investigate the effects of down-regulated HLA-G on extravillous trophoblasts. The short hairpin RNA (shRNA) vector targeting HLA-G was constructed and transfected into the human first-trimester extravillous trophoblast cell line TEV-1. Western blotting and reverse transcription polymerase chain reaction (RT-PCR) revealed that in HLA-G shRNA transfected cells, the expression of HLA-G was significantly decreased. HLA-G expression was also visualised by confocal imaging. The HLA phenotype of TEV-1 cells and inhibitory receptors expression in NK cells were analysed by flow cytometry. A comparison between HLA-G knockdown and non-knockdown cells showed a significant difference in the HLA expression profile without altering HLA-C and HLA-E. Both primary NK cells and NK-92 cell line exhibited potent cytotoxicity against HLA-G knockdown cells via standard 4-h Cr-51 release assays. Expression of ILT2, ILT4 and KIR2DL4 in NK cells was unchanged after 4 h of co-culture, while KIR2DL4 expression increased after 48 h. We conclude that HLA-G contributes to trophoblast/NK interaction, acting as a key regulator of NK cytolysis in this human extravillous trophoblast cell model. In addition, TEV-1 cells share common HLA phenotype characters with extravillous trophoblast cells, and thus might be used as a good cell model. HLA-C expression in trophoblasts is not correlated with HLA-G translation and HLA-C alone was sufficient to boost HLA-E surface expression. In addition, RNA interference could be employed as a feasible and effective method to study HLA-G functions. (C) 2010 Elsevier Ltd. All rights reserved.