MicroRNA-210调节Treg细胞在子痫前期免疫发病机制的研究
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摘要
目的评估子痫前期患者调节性T细胞(Treg)数量,探讨子痫前期中microRNA-210(miR-210)、叉头样转录因子p3(Foxp3)基因表达水平,进而揭示miR-210与Foxp3在子痫前期中的调控机制。方法采用酶联免疫吸附法检测子痫前期29例、正常孕妇27例及健康非妊娠妇女20例血清细胞因子(IL-6、IL-10、IL-17、TGF-β1)的表达水平。采用流式细胞术分析外周血CD4~+CD25~+CD127low/-细胞。采用实时荧光定量PCR(qPCR)方法检测子痫前期患者和正常孕妇胎盘组织中Foxp3和维甲酸相关孤儿受体C(RORc)、miR-210的表达水平。蛋白质印迹法检测胎盘中Foxp3蛋白的表达。结果⑴子痫前期患者血清IL-6、IL-17和TGF-β1浓度较正常孕妇明显升高,Treg细胞因子IL-10水平低于正常孕妇(P <0. 05)。⑵子痫前期患者外周血CD4~+CD25~+CD127-/CD4~+T细胞百分率明显低于正常妊娠组及健康非妊娠妇女(P <0. 001)。⑶子痫前期患者胎盘组织中表现为Foxp3 mRNA水平明显低于正常孕妇,子痫前期患者的RORc mRNA水平明显高于正常孕妇,miRNA-210在子痫前期患者组的表达增强(P <0. 01)。⑷子痫前期患者胎盘组织Foxp3蛋白表达降低(P <0. 05)。结论子痫前期患者存在Treg细胞数量减少和Treg/Th17失衡,这些变化破坏母体对胎儿的免疫耐受。在子痫前期患者胎盘组织中Foxp3表达水平明显降低,且Foxp3的表达与miR-210的表达水平有关。
Objective The aim of this study was to evaluate the number of Treg cells in preeclampsia(PE) patients,to explore the expression levels of microRNA-210(microRNA-210) and forkhead box p3(Foxp3) genes in preeclampsia,and to reveal the regulatory mechanism of microRNA-210 and Foxp3 in preeclampsia. Methods Serum levels of cytokines [interleukin(IL)-6,IL-10,IL-17,and transforming growth factor-beta 1(TGF-β1)]were detected with enzyme-linked immunosorbent assay(ELISA). 29 patients with late-onset preeclampsia(≥36 weeks of gestation),27 pregnant women with normal uncomplicated pregnancies(≥36 weeks of gestation) and 20 healthy non-pregnant women were enrolled in the study.Reverse-transcription polymerase chain reaction(qRT-PCR) was performed to detect mRNA expression for maternal placenta retinoic acid-related orphan receptor C(RORc),Foxp3,and miR-210. Foxp3 protein expression was evaluated by Western blot. Results ⑴ The serum levels of IL-6,IL-17 and TGF-beta 1 in preeclampsia patients were significantly higher than those in normal pregnant women,and the level of Treg cytokine IL-10 was lower than that in normal pregnant women(P < 0. 05). ⑵ The percentage of CD4~+CD25~+CD127-/CD4~+T cells in peripheral blood of preeclampsia patients was significantly lower than that of normal pregnancy group and healthy non-pregnant women(P < 0. 001). ⑶ The mRNA expression of Foxp3 in placenta of preeclampsia patients was significantly lower than that of normal pregnant women,RORc in preeclampsia patients was significantly higher than that of normal pregnant women,and the expression of microRNA210 in preeclampsia patients was enhanced(P < 0. 01). ⑷ Consistent with mRNA expression results,lower protein expression levels of Foxp3 was observed in patients with PE compared with normal pregnant subjects. Conclusions Treg cells decreased in preeclampsia patients and Treg/Th17 imbalance existed in preeclampsia patients,which regulate maternal immune tolerance to fetuses. The expression of Foxp3 in placenta of preeclampsia patients was significantly decreased,which was correlated with the expression of microRNA-210.
Objective The aim of this study was to evaluate the number of Treg cells in preeclampsia(PE) patients,to explore the expression levels of microRNA-210(microRNA-210) and forkhead box p3(Foxp3) genes in preeclampsia,and to reveal the regulatory mechanism of microRNA-210 and Foxp3 in preeclampsia. Methods Serum levels of cytokines [interleukin(IL)-6,IL-10,IL-17,and transforming growth factor-beta 1(TGF-β1)]were detected with enzyme-linked immunosorbent assay(ELISA). 29 patients with late-onset preeclampsia(≥36 weeks of gestation),27 pregnant women with normal uncomplicated pregnancies(≥36 weeks of gestation) and 20 healthy non-pregnant women were enrolled in the study.Reverse-transcription polymerase chain reaction(qRT-PCR) was performed to detect mRNA expression for maternal placenta retinoic acid-related orphan receptor C(RORc),Foxp3,and miR-210. Foxp3 protein expression was evaluated by Western blot. Results ⑴ The serum levels of IL-6,IL-17 and TGF-beta 1 in preeclampsia patients were significantly higher than those in normal pregnant women,and the level of Treg cytokine IL-10 was lower than that in normal pregnant women(P < 0. 05). ⑵ The percentage of CD4~+CD25~+CD127-/CD4~+T cells in peripheral blood of preeclampsia patients was significantly lower than that of normal pregnancy group and healthy non-pregnant women(P < 0. 001). ⑶ The mRNA expression of Foxp3 in placenta of preeclampsia patients was significantly lower than that of normal pregnant women,RORc in preeclampsia patients was significantly higher than that of normal pregnant women,and the expression of microRNA210 in preeclampsia patients was enhanced(P < 0. 01). ⑷ Consistent with mRNA expression results,lower protein expression levels of Foxp3 was observed in patients with PE compared with normal pregnant subjects. Conclusions Treg cells decreased in preeclampsia patients and Treg/Th17 imbalance existed in preeclampsia patients,which regulate maternal immune tolerance to fetuses. The expression of Foxp3 in placenta of preeclampsia patients was significantly decreased,which was correlated with the expression of microRNA-210.
引文
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[16]Zhao M,Wang LT,Liang GP,et al.Up regulation of microRNA210 induces immune dysfunction via targeting FOXP3 in CD4+Tcells of psoriasis vulgaris[J].Clin Immunol,2014,150(1):22-30.DOI:10.1016/j.clim.2013.10.009.
[17]Rouas R,Fayyad-Kazan H,El Zein N,et al.Human natural Treg microRNA signature:role of microRNA-31 and microRNA-21 in FOXP3 expression[J].Eur J Immunol,2009,39(6):1608-1618.DOI:10.1002/eji.200838509.
[18]Zhou X,Jeker LT,Fife BT,et al.Selective miRNA disruption in T reg cells leads to uncontrolled autoimmunity[J].TJ Exp Med,2008,205(9):1983-1991.DOI:10.1084/jem.20080707.
[2]Ilekis JV,Reddy UM,Roberts JM,et al.Preeclampsia A pressing problem:An executive summary of a national institute of child health and human development workshop[J].Reprod Sci,2007,14(6):508-523.DOI:10.1177/1933719107306232.
[3]Lok CA,Jebbink J,Nieuwland R,et al.Leukocyte activation and circulating leukocyte derived microparticles in preeclampsia[J].Am J Reprod Immunol,2009,61(5):346-359.DOI:10.1111/j.1600-0897.2009.00701.x.
[4]Sakaguchi S,Sakaguchi N,Asano M,et al.Immunologic self tolerance maintained by activated T cells expressing IL 2 receptor alpha chains(CD25).Breakdown of a single mechanism of self tolerance causes various autoimmune diseases[J].J Immunol,1995,155(3):1151-1164.
[5]Xiong YH,Yuan Z,He L,et al.Effects of estrogen on CD4+CD25+regulatory T cell in peripheral blood during pregnancy[J].Asian Pac J Trop Med,2013,6(9):748-752.DOI:10.1016/S1995-7645(13)60131-5.
[6]Koucky'M,MalícˇkováK,Cindrová-Davies T,et al.Low levels of circulating T-regulatory lymphocytes and short cervical length are associated with preterm labor[J].J Reprod Immunol,2014,106:110-117.DOI:10.1016/j.jri.2014.04.001.
[7]Sakaguchi S,Ono M,Setoguchi R,et al.Foxp3+CD25+CD4+natural regulatory T cells in dominant self tolerance and autoimmune disease[J].Immunol Rev,2006,212:8-27.DOI:10.1111/j.0105-2896.2006.00427.x.
[8]Figueiredo AS,Schumacher A.The T helper type 17/regulatory Tcell paradigm in pregnancy[J].Immunology,2016,148(1):13-21.DOI:10.1111/imm.12595.
[9]Darmochwal Kolarz D,Kludka Sternik M,Tabarkiewicz J,et al.The predominance of Th17 lymphocytes and decreased number and function of Treg cells in preeclampsia[J].J Reprod Immunol,2012,93(2):75-81.DOI:10.1016/j.jri.2012.01.006.
[10]Santner-Nanan B,Peek MJ,Khanam R,et al.Systemic increase in the ratio between Foxp3+and IL 17 producing CD4+T cells in healthy pregnancy but not in preeclampsia[J].J Immunol,2009,183(11):7023-7030.DOI:10.4049/jimmunol.0901154.
[11]Huntzinger E,Izaurralde E.Gene silencing by microRNAs:Contributions of translational repression and mRNA decay[J].Nat Rev Genet,2011,12(2):99-110.DOI:10.1016/j.cellimm.2008.04.008.
[12]Morales-Prieto DM,Ospina-Prieto S,Chaiwangyen W,et al.Pregnancy-associated miRNA-clusters[J].J Reprod Immunol,2013,97(1):51-61.DOI:10.1016/j.jri.2012.11.001.
[13]Lin Z,Murtaza I,Wang K,et al.miR 23a functions downstream of NFATc3 to regulate cardiac hypertrophy[J].Proc Natl Acad Sci U S A,2009,106(29):12103-12108.DOI:10.1073/pnas.0811371106.
[14]Shen XC,Lu Y,Qian ZY.Effects of crocetin on the matrix metalloproteinases in cardiac hypertrophy induced by norepinephrine in rats[J].J Asian Nat Prod Res,2006,8(3):201-208.DOI:10.1080/10286020412331286452.
[15]Mouillet JF,Chu T,Hubel CA,et al.The levels of hypoxia-regulated microRNAs in plasma of pregnant women with fetal growth restriction[J].Placenta,2010,31(9):781-784.DOI:10.1016/j.placenta.2010.07.001.
[16]Zhao M,Wang LT,Liang GP,et al.Up regulation of microRNA210 induces immune dysfunction via targeting FOXP3 in CD4+Tcells of psoriasis vulgaris[J].Clin Immunol,2014,150(1):22-30.DOI:10.1016/j.clim.2013.10.009.
[17]Rouas R,Fayyad-Kazan H,El Zein N,et al.Human natural Treg microRNA signature:role of microRNA-31 and microRNA-21 in FOXP3 expression[J].Eur J Immunol,2009,39(6):1608-1618.DOI:10.1002/eji.200838509.
[18]Zhou X,Jeker LT,Fife BT,et al.Selective miRNA disruption in T reg cells leads to uncontrolled autoimmunity[J].TJ Exp Med,2008,205(9):1983-1991.DOI:10.1084/jem.20080707.