Dai Lan, Gu Li-ying, Zhu Jie, Shi Jun, Wang Yao, Ji Fang, Di Wen
Department of Obstetrics and Gynecology, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Zhonghua Fu Chan Ke Za Zhi. 2011 Nov;46(11):817-21.
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.
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.
(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).
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.
研究微小RNA 199a（miR-199a）对子宫内膜异位症患者在位子宫内膜基质细胞（ESC）黏附、迁移及侵袭能力的调控作用。
采用脂质体2000将miR-199a模拟物或阴性对照（NC）RNA转染至ESC。分别通过细胞黏附实验、划痕实验、细胞迁移实验及基质胶侵袭实验检测ESC的黏附、迁移及侵袭能力。采用荧光素酶报告基因实验评估IKKβ是否为miR-199a的靶基因。通过蛋白质免疫印迹法检测IKKβ、抑制性κBα（IκB-α）、磷酸化IκB-α（p-IκB-α）及核因子κB（NF-κB）蛋白的表达。
（1）黏附能力：miR-199a组的黏附抑制率为（14±4）%，对照组为0，差异有统计学意义（P<0.01）。（2）迁移和侵袭能力：在划痕实验中，转染miR-199a的ESC的划痕愈合率低于对照组。在迁移和侵袭实验中，miR-199a组的迁移和侵袭能力与NC组相比显著降低[分别为130±31比247±36（P<0.01）；63±15比133±17（P<0.01）]。（3）miR-199a组的荧光素酶活性显著低于对照组[0.160±0.006比0.383±0.083（P<0.01）]。miR-199a组的IKKβ、p-IκB-α、IκB-α及NF-κB蛋白水平分别为0.350±0.195、0.443±0.
