miR-30e 通过靶向 PRF1 调节 NK 细胞活性对母胎界面免疫耐受的影响。
Effect of miR-30e regulating NK cell activities on immune tolerance of maternal-fetal interface by targeting PRF1.
机构信息
Department of Obstetrics and Gynecology, Renmin Hosptial of Wuhan University, Wuhan 430060, PR China.
Reproductive Medical Center, Renmin Hosptial of Wuhan University, Wuhan 430060, PR China.
出版信息
Biomed Pharmacother. 2019 Jan;109:1478-1487. doi: 10.1016/j.biopha.2018.09.172. Epub 2018 Nov 13.
AIM
Natural killer (NK) cells, as key regulatory cells, accumulate at the maternal-fetal interface in large numbers. This study explored the effect of miR-30e on regulating the activity and function of peripheral blood NK cells (PB-NK cells) and decidua NK cells (D-NK cells) by targeting PRF1 in immune tolerance of maternal-fetal interface.
METHODS
Expressions of miR-30e in PB and decidua tissues from 49 patients with recurrent spontaneous abortion and 52 normal pregnant women were measured using PCR. NK cells were isolated from PB and decidua tissues and identified by flow cytometry (FCM). In PB-NK cells and D-NK cells activated by IFN-α, expressions of miR-30e and PRF1 were determined by PCR and Western blot. Negative controls of miR-30e mimics/inhibitors and siRNA against PRF1 were transfected in PB-NK cells and D-NK cells. Expressions of miR-30e and PRF1 were determined and their relationship was verified. Expressions of KIR2DL1, NKp44, IFN-γ, TNF-α, IL-4 and IL-10 were determined by FCM. Cytotoxicity kit was used to identify the cytotoxicity of NK cells. PCR and ELISA were employed to measure expression of VEGF, Ang-2 and PGF in D-NK cells.
RESULTS
After activation by IFN-α, D-NK cells and PB-NK cells showed decreased miR-30e expression and increased PRF1 expression in normal non-pregnant women. PRF1 is a target gene of miR-30e and miR-30e negatively regulated PRF1 expression. The treatment of miR-30e mimics elevated KIR2DL1 expression and decreased NKp44 expression in PB-NK or D-NK cells. Moreover, up-regulation of miR-30e expression suppressed cytotoxicity, corresponding to increased expression of IL-4and IL-10 and reduced expression of IFN-γ and TNF-α in PB-NK and D-NK cells, as well as enhanced expression of VEGF, Ang-2 and PGF in D-NK cells. Transfection of miR-30e inhibitors could reverse the tendencies.
CONCLUSION
Up-regulated miR-30e can reduce the cytotoxicity of PB-NK cells and D-NK cells by targeting PRF1, whereby inhibiting Th1 tolerance phenotype and inducing Th2 immunodominance. miR-30e may be contributive to creating a micro-immune tolerance environment of maternal-fetal interface.
目的
自然杀伤 (NK) 细胞作为关键的调节细胞，在母胎界面大量积聚。本研究通过靶向 PRF1 探讨 miR-30e 对调节母胎界面免疫耐受中外周血 NK 细胞 (PB-NK 细胞) 和蜕膜 NK 细胞 (D-NK 细胞) 活性和功能的影响。
方法
采用 PCR 法检测 49 例复发性自然流产患者和 52 例正常孕妇 PB 和蜕膜组织中 miR-30e 的表达。采用流式细胞术 (FCM) 分离 PB 和蜕膜组织中的 NK 细胞并进行鉴定。在 IFN-α 激活的 PB-NK 细胞和 D-NK 细胞中，采用 PCR 和 Western blot 法检测 miR-30e 和 PRF1 的表达。转染 PB-NK 细胞和 D-NK 细胞的 miR-30e 模拟物/抑制剂的阴性对照和针对 PRF1 的 siRNA。检测 miR-30e 和 PRF1 的表达，并验证其相关性。采用 FCM 法检测 KIR2DL1、NKp44、IFN-γ、TNF-α、IL-4 和 IL-10 的表达。采用细胞毒性试剂盒鉴定 NK 细胞的细胞毒性。采用 PCR 和 ELISA 法检测 D-NK 细胞中 VEGF、Ang-2 和 PGF 的表达。
结果
在 IFN-α 激活后，正常未妊娠妇女的 D-NK 细胞和 PB-NK 细胞表现出 miR-30e 表达降低和 PRF1 表达升高。PRF1 是 miR-30e 的靶基因，miR-30e 负调控 PRF1 的表达。miR-30e 模拟物处理可上调 PB-NK 或 D-NK 细胞中的 KIR2DL1 表达，下调 NKp44 表达。此外，miR-30e 表达上调抑制细胞毒性，导致 PB-NK 和 D-NK 细胞中 IL-4 和 IL-10 表达增加，IFN-γ 和 TNF-α 表达减少，D-NK 细胞中 VEGF、Ang-2 和 PGF 表达增强。转染 miR-30e 抑制剂可逆转这些趋势。
结论
上调的 miR-30e 通过靶向 PRF1 降低 PB-NK 细胞和 D-NK 细胞的细胞毒性，从而抑制 Th1 耐受表型并诱导 Th2 免疫优势。miR-30e 可能有助于形成母胎界面的微免疫耐受环境。
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