微小RNA-199a对子宫内膜间质细胞黏附、迁移和侵袭能力的调控作用

目的 探讨微小RNA-199a (miR-199a)对子宫内膜异位症(内异症)患者在位子宫内膜间质细胞黏附、迁移和侵袭能力的调控作用.方法 利用脂质体lipofectamine 2000将成熟miR-199a模拟物(转染组)和阴性对照物(对照组)转染人子宫内膜间质细胞.采用黏附试验检测转染后细胞的体外黏附能力,划痕试验和迁移实验检测转染后细胞的迁移能力,重组细胞基底膜侵袭实验检测细胞的侵袭能力.运用荧光素酶报告基因试验验证核因子κB(NF-κB)抑制蛋白激酶β(IKKβ)是miR-199a的靶基因.应用蛋白印迹法检测IKKβ、NF-κB抑制蛋白α(IκB-α)、磷酸化IκB-α( p-IκB-α)和NF-κB蛋白的表达.结果 (1)细胞黏附能力:转染组细胞的黏附抑制率为(14±4)％,明显高于对照组细胞(0),差异有统计学意义(P＜0.01).(2)细胞迁移和侵袭能力:划痕试验显示,转染组细胞48 h后,划痕愈合程度低于对照组;迁移试验显示,转染组和对照组穿膜细胞数分别为(130 ±31)、(247±36)个/高倍视野(×200),两组比较,差异有统计学意义(P＜0.01);侵袭试验显示,两组穿透基底膜细胞数分别为(63±15)、(133±17)个/高倍镜,两组比较,差异有统计学意义(P＜0.01).(3)荧光素酶的表达水平:荧光素酶报告基因试验显示,转染组和对照组细胞的荧光素酶表达水平分别为0.160±0.006和0.383±0.083.两组比较,差异有统计学意义(P＜0.01).(4)蛋白表达水平:与对照组细胞蛋白表达水平(设定为1.000)比较,转染组细胞中IKKβ、p-IκB-α、IκB-α和NF-κB蛋白的表达水平分别为0.350±0.195、0.443±0.076、1.970±0.486和0.454±0.147,两组比较,差异均有统计学意义(P＜0.01).结论 miR-199a能抑制人子宫内膜间质细胞的黏附、迁移和侵袭能力;IKKβ为miR-199a的靶基因;miR-199a抑制内膜间质细胞侵袭能力的机制之一可能是通过调控靶基因IKKβ,抑制NF-κB通路的活化水平.

Regulation of microRNA-199a on adhesion, migration and invasion ability of human endometrial stromal cells

Objective To study the regulation of microRNA 199a (miR-199a) on adhesion,migration and invasion ability of human eutopic endometrial stromal cells (ESC) from patients with endometriosis.Methods ESC were transfected with miR-199a mimics or negative control (NC) RNA by lipofectamine 2000.The adhesion,migration and invasion ability of ESC were detected by cell adhesion assay,scratch assay,cell migration assay and matrigel invasion assay,respectively.Luciferase reporter assay was used to evaluate whether IKKβ was the target gene of miR-199a.The expression of ikappa B kinase beta (IKKβ),inhibitory kappa B alpha (IκB-α),phospho-IκB-α (p-IκB-α) and nuclear factor-kappa B (NF κB) protein were measured by western blot.Results ( 1 ) Adhesion potential:the adhesion inhibitory rates were ( 14 ± 4 )％ in miR-199a group and 0 in control group,which showed significant difference (P＜0.01 ).(2) Migration and invasion:in the scratch assay,ESC transfected with miR-199a exhibited a lower scratch closure rate than that of controls.In migration and invasion assays,the migration and invasion ability of miR-199a group were significantly decreased compared with those of NC group [ 130 ± 31 vs.247±36 (P＜0.01); 63 ± 15 vs.133 ± 17 (P＜0.01),respectively].(3) The luciferase activity of miR-199a group was significantly lowered than that of control group [ 0.160 ± 0.006 vs.0.383 ± 0.083 ( P ＜0.01 ) ].The protein levels of IKKβ,p-IκB-α,IκB-α and NF-κB of 0.350 ±0.195,0.443 ±0.076,1.970 ±0.486 and 0.454 ± 0.147 in miR-199a group were significantly different compared with the NC group in which the protein levels were set at 1.000 ( P ＜ 0.01 ).Conclusions miR-199a can inhibit the adhesion,migration and invasion of the ESC.IKKβ is the target gene of miR-199a in ESC.One of the mechanisms of the inhibition effect is probably that miR-199a inhibits the activation of NF-κB signaling pathway by targeting IKKβ gene.