MiR-199a attenuates endometrial stromal cell invasiveness through suppression of the IKKβ/NF-κB pathway and reduced interleukin-8 expression

Abstract

MicroRNAs have recently been identified as regulators that modulate target gene expression and are suggested to be involved in the development and progression of endometriosis. This study was undertaken to analyze the expression level of microRNA-199a (miR-199a) in paired ovarian endometrioma and eutopic endometrium from women with endometriosis, and to investigate the contribution of miR-199a to the invasive capability of endometrial stromal cells (ESCs). Cell adhesion, migration and Matrigel invasion assays were carried out to measure the invasiveness of ESCs. Bioinformatics prediction, reporter gene assay, PCR, western blotting and ELISA were performed to identify miR-199a targets and related signaling pathways. The results showed that the expression level of miR-199a was lower in the eutopic endometrium from women with endometriosis, and even lower in the paired ovarian endometrioma, compared with the expression in normal controls. Moreover, ectopic expression of miR-199a attenuated ESC adhesion, migration and invasiveness. MiR-199a targeted and inhibited IkappaB kinase beta (IKKβ) in ESCs. Accompanied by IKKβ reduction, miR-199a suppressed nuclear factor-kappa B (NF-κB) pathway activation and interleukin-8 (IL-8) production in ESCs. All these findings suggest that miR-199a, down-regulated in endometriosis, attenuates the invasive capability of ESCs in vitro partly through IKK/NF-κB pathway suppression and reduced IL-8 expression. In conclusion, miR-199a could be involved in the pathogenesis of endometriosis.

Figure 1

The relative expression levels of miR-199a in 12 paired eutopic endometrium and ovarian endometrioma from patients with endometriosis and in 12 endometrium from women without endometriosis (control endometrium). Data are expressed as mean ± SD miR-199a expression is presented as fold change relative to the women without endometriosis group (control endometrium = 1). Mann–Whitney U-test was used to draw comparisons between endometrial tissues from controls and endometriosis patients; while Wilcoxon signed-ranks test was used to draw comparisons between paired ovarian endometrioma and eutopic endometrium from endometriosis patients.

Figure 2

MiR-199a suppressed ESC invasiveness. (a) qRT-PCR analysis of miR-199a in ESCs at 24 h post-transfection. (b) Cell adhesion assay. At 48 h post-transfection, cells were added to a precoated 96-well plate containing Matrigel and allowed to adhere for 1 h at 37°C. The number of attached cells was counted under a microscope at ×200 magnification. (c) Cell migration and invasion assay. (c1) Representative hematoxylin and eosin staining fields of migrated or invaded cells on the membrane (c2) Average number of the migrated or invaded cells from triplicate cultures. The results are shown as the mean ± SD from triplicate cultures. (*P<0.05 versus NC group).

Figure 3

MiR-199a targets and inhibits IKKβ in ESCs. (a) The alignment between hsa-miR-199a and the 3′UTR of IKKβ, identified by TargetScan software. (b) The miR-199a target region, or a mutant, was cloned into the 3′UTR of a luciferase gene. These constructs were introduced into ESCs, in addition to miR-199a mimics or NC. After 48 h, luciferase activity was measured, averaged and plotted. The y-axis represents the renilla luciferase activity normalized to firefly luciferase activity. (*P<0.05 versus the other three groups) (c) The IKKβ mRNA level in transfected ESCs was analyzed by qRT-PCR at 24 h post-transfection. (d) The IKKβ protein level in transfected ESCs was analyzed by western blot at 48 h post-transfection. The results are shown as the mean ± SD from triplicate cultures. (*P<0.05 versus NC group).

Figure 4

MiR-199a inhibits NF-κB cellular localization, IκB-α phosphorylation and NF-κB pathway activation in ESCs. ESCs were transfected with miR-199a mimics or NC for 48 h. (a) Cells were fixed and stained for the p65 subunit of NF-κB (red, p65; blue, nuclei). Arrows indicate NF-κB p65 immunostaining in the cell nucleus, and asterisks indicate NF-κB p65 in the cytoplasm. (b) Summarized data show the immunofluorescent staining intensity of p65 in the nuclear area of ESCs transfected with NC or miR-199a. (*P<0.05 versus NC group) (c) ESCs were lysed after transfection, and nuclear extracts were prepared and immunoblotted for either the p65 subunit of NF-κB or nucleolin. Whole cell lysates of transfected ESCs were immunoblotted for phospho-IκB-α, total IκB-α and GAPDH. Nucleolin and GAPDH were used as the loading control. (d) Summarized data show the average protein of NF-κB p65, phospho-IκB-α and total IκB-α (normalized to the protein level of GAPDH or nucleolin) corresponding to the bands shown in the western blots. The results are shown as the mean ± SD from triplicate cultures. (*P<0.05 versus NC group).

Figure 5

MiR-199a represses the IL-8 production from ESCs. (a) ESCs were transfected with miR-199a mimics or NC for 24 h, and IL-8 mRNA expression was analyzed by qRT-PCR. (b) ESCs were transfected with miR-199a mimics or NC for 48 h, and IL-8 protein secretion in the culture medium was analyzed by ELISA. Results are presented as mean ± SD from three separate experiments (*P<0.05 versus NC group).

Figure 6

Overview of the negative regulation of the IKK/NF-κB pathway by miR-199a. ESCs have high levels of IKKβ expression, and when stimulated, NF-κB activation leads to cytokine production and cell invasion. While miR-199a was transfected into ESCs, it used the inhibitory machinery to reduce the expression of its target gene IKKβ, thereby suppressing the IKK/NF-κB pathway activation and attenuating cytokine production (for example IL-8) and cell invasiveness.