miRNA-146调控TLR4/NF-κB通路促进滋养层细胞增殖和迁移

miRNA-146promotes trophoblast proliferation and migration by regulating TLR4/NF-κB pathway

目的探讨miRNA-146是否通过调控Toll样受体4/核因子-κB(TLR4/NF-κB)信号通路促进滋养层细胞HTR-8/SVneo增殖和迁移,以期为不明原因复发性流产(uRSA)的发病机制研究和防治提供参考。方法(1)细胞研究:将转染后的胎盘滋养层细胞HTR-8/SVneo分成miRNA-146抑制剂组、抑制剂对照组、miRNA-146模拟物组、模拟物对照组,检测各组HTR-8/SVneo细胞生长的活力、增殖、凋亡、迁移情况,通过实时荧光定量PCR(qRT-PCR)和Western blot检测各组TLR4、NF-κB、肿瘤坏死因子(TNF-α)和白介素-6(IL-6)的mRNA和蛋白相对表达水平。(2)动物研究:建立Dicer酶敲除的uRSA小鼠模型,将正常妊娠小鼠作为对照组,通过qRT-PCR检测胎盘组织的miRNA-146 mRNA表达情况,采用免疫组织化学法检测胎盘组织的TLR4、NF-κB、TNF-α、IL-6蛋白表达情况。结果(1)细胞研究:与模拟物对照组比较,miRNA-146模拟物组HTR-8/SVneo细胞克隆形成率、增殖能力及迁移能力显著增加(P<0.05),细胞凋亡率显著降低(P<0.05);与抑制剂对照组和miRNA-146模拟物组比较,miRNA-146抑制剂组HTR-8/SVneo细胞克隆形成率、增殖能力及迁移能力显著降低(P<0.05),而细胞凋亡率显著增加(P<0.05)。与模拟物对照组比较,miRNA-146模拟物组的TLR4、NF-κB、TNF-α、IL-6蛋白和mRNA相对表达量显著降低(P<0.05);与抑制剂对照组和miRNA-146模拟物组比较,miRNA-146抑制剂组的TLR4、NF-κB、TNF-α、IL-6蛋白和mRNA相对表达量显著增加(P<0.05)。不同浓度(10~80μmol/L)miRNA-146对HTR-8/SVneo细胞生长活力的增长均有显著影响(P<0.05),并呈浓度依赖性。(2)动物研究:与对照组比较,uRSA组小鼠胎盘组织中miRNA-146 mRNA的相对表达量显著降低[(0.32±0.19)vs.(0.96±0.24),t=12.473,P<0.05],而TLR4、NF-κB、TNF-α、IL-6的蛋白表达有增高趋势。结论上调miRNA-146的表达具有促进胎盘滋养层细胞的增殖和迁移作用,其机制可能与负性调控TLR4/NF-κB通路相关。miRNA-146有望成为防治uRSA的潜在靶标。

Objective:To investigate whether micro RNA-146(miRNA-146)regulates signaling pathway of Toll like receptor 4/nuclear factor-κB(TLR4/NF-κB)to promote the proliferation and migration of HTR-8/SVneo trophoblast cells,and provide reference for the pathogenesis and prevention of unexplained recurrent spontaneous abortion(uRSA).Methods:(1)The transfected HTR-8/SVneo placental trophoblast cells were divided into the miRNA-146 inhibitor group,inhibitor control group,miRNA-146mimic group and mimic control group.The vitality,proliferation,apoptosis and migration of HTR-8/SVneo cells in each group were detected.The quantitativereal-time PCR(qRT PCR)and Western blot were used to detect the mRNA and protein expression ofmiRNA-146,TLR4,NF-κB,tumor necrosis factor alpha(TNF-α)and interleukin-6(IL-6).(2)The Dicerenzyme knockout uRSA mouse model was established,and the normal pregnant mice was collected as thecontrol group.The miRNA-146mRNA expression in placental tissue was detected by qRT-PCR.Theprotein expressions of TLR4,NF-κB,TNF-αand IL-6 in placental tissue were detected byimmunohistochemical assay.Results:(1)Compared with the mimic control group,the clone formation rate,proliferation ability,andmigration ability of HTR-8/SVneo cells in the miRNA-146mimic group were significantly increased(P<0.05),while apoptosis rate was significantly reduced in the miRNA-146 mimic group(P<0.05).Compared with the inhibitor control group and the miRNA-146mimic group,the clone formation rate,proliferation ability and migration ability of HTR-8/SVneo cells in the miRNA-146inhibitor group weresignificantly reduced(P<0.05),while the apoptosis rate was significantly increased in the miRNA-146inhibitor group(P<0.05).Compared with the mimic control group,the protein and mRNA relativeexpression levels of TLR4,NF-κB,TNF-αand IL-6were significantly reduced in miRNA-146mimic group(P<0.05).Compared with the inhibitor control group and the miRNA-146mimic group,the protein andmRNA relative expression levels of TLR4,NF-κB,TNF-αand IL-6were significantly increased in miRNA-146inhibitor group(P<0.05).Different concentrations(10-80μmol/L)of miRNA-146significantly affectedthe growth and vitality of HTR-8/SVneo cells(P<0.05),and showed a dose-dependent manner.(2)Comparedwith the control group,the relative mRNA expression levels of miRNA-146in mice placental tissue ofuRSA group were significantly decreased[(0.32±0.19)vs.(0.96±0.24),t=12.473,P<0.05],while therelative protein expression levels of TLR4,NF-κB,TNF-αand IL-6showed an increasing trend.Conclusions:Up-regulation of miRNA-146expression can promote the proliferation and migration oftrophoblast cells,and the mechanism may be related to negative regulation of TLR4/NF-κB pathway.The miRNA-146might be expected to become a potential indicator for the prevention and treatment of uRSA.