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. 2017 Sep;24(9):1264-1274.
doi: 10.1177/1933719116682877. Epub 2016 Dec 21.

miR-181b-5p Modulates Cell Migratory Proteins, Tissue Inhibitor of Metalloproteinase 3, and Annexin A2 During In Vitro Decidualization in a Human Endometrial Stromal Cell Line

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miR-181b-5p Modulates Cell Migratory Proteins, Tissue Inhibitor of Metalloproteinase 3, and Annexin A2 During In Vitro Decidualization in a Human Endometrial Stromal Cell Line

Amanda Graham et al. Reprod Sci. .
Free PMC article

Abstract

Decidualization is essential for successful embryo implantation and is regulated by concerted actions of growth factors and hormones. More recently, microRNAs, small RNA molecules that regulate posttranscriptional gene expression, have been implicated to play a role in the decidualization process. Of these microRNAs, miR-181b-5p has been associated with decidualization but its precise role and targets are not well established. To address this gap in our knowledge, we assessed the expression of miR-181b-5p, and its target tissue inhibitor of metalloproteinase 3 (TIMP-3), during in vitro decidualization using the well-characterized human endometrial stromal cell line, t-HESC. miR-181b-5p expression was highest prior to decidualization and significantly decreased in response to decidualization stimulus. In contrast, TIMP-3 expression was absent prior to in vitro decidualization and increased during decidualization. Regulation of TIMP-3 expression by miR-181b-5p was confirmed in vitro by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot analysis, and 3' untranslated region reporter constructs. To identify unforeseen targets of miR-181b-5p during in vitro decidualization, t-HESC cells were transfected with pre- miR-181b-5p, and protein profiles were determined by 2-dimensional differential in-gel electrophoresis followed by matrix-assisted laser desorption-ionization time-of-flight/time-of-flight (MALDI TOF/TOF) tandem mass spectrometry. Of these proteins, several downregulated proteins associated with cell migration were identified including annexin A2, which we subsequently confirmed by qRT-PCR and Western blot analysis to be regulated by miR-181b-5p. In conclusion, miR-181b-5p is downregulated during the process of in vitro decidualization and may regulate the expression of proteins associated with cell migration including TIMP-3 and annexin A2.

Keywords: decidualization; endometrium; miR-181b-5p; miRNA.

Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Human endometrial stromal cell line, t-HESC miR-181b-5p expression during in vitro decidualization. miR-181b-5p levels were quantitated as described in “Materials and Methods” by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and normalized to U58 levels. Data are displayed as the mean ± standard error of the mean (SEM) and are representative of 4 independent samples per time point per treatment (n = 4). Data were analyzed by 1-way analysis of variance (ANOVA) for comparison across time points and expressed as fold change from day 0 (not decidualized) values. When an F test indicated statistical significance, post hoc analysis was made using the Tukey honest significant difference (HSD) procedure. Significance was set at P < .05 for all comparisons. Different letters indicate statistical significance as determined by 1-way ANOVA.
Figure 2.
Figure 2.
Human endometrial stromal cell line, t-HESC tissue inhibitor of metalloproteinase 3 (TIMP-3) messenger RNA (mRNA) expression during in vitro decidualization. TIMP-3 mRNA levels were quantitated as described in “Materials and Methods” by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and normalized to 18S ribosomal RNA (rRNA) levels using RNA from the same samples analyzed for miR-181b-5p (Figure 1). Data are displayed as the mean ± standard error of the mean (SEM) and are representative of 4 independent samples per time point per treatment (n = 4). Data were analyzed by 1-way analysis of variance (ANOVA) for comparison across time points and expressed as fold change from day 0 (not decidualized) values. When an F test indicated statistical significance, post hoc analysis was made using the Tukey honest significant difference (HSD) procedure. Significance was set at P < .05 for all comparisons. Different letters indicate statistical significance as determined by 1-way ANOVA.
Figure 3.
Figure 3.
Human endometrial stromal cell line, t-HESC tissue inhibitor of metalloproteinase 3 (TIMP-3) protein expression increases during in vitro decidualization. A, TIMP-3 protein expression was analyzed by Western analysis and normalized to β-actin as described in “Materials and Methods.” B, Data are displayed as the mean ± standard error of the mean (SEM) for TIMP-3/β-actin for 4 independent observations (n = 4). Data were analyzed by 1-way analysis of variance (ANOVA) for comparison across days of in vitro decidualization and are compared to day 0 values. When an F test indicated statistical significance, post hoc analysis was made using the Tukey honest significant difference (HSD) procedure. Significance was set at P < .05 for all comparisons. Different letters indicate statistical significance as determined by 1-way ANOVA
Figure 4.
Figure 4.
miR-181b-5p forced expression reduces t-HESC TIMP-3 messenger RNA (mRNA) expression but does not affect in vitro decidualization-associated prolactin expression. t-HESC cells were cultured and transfected with either a nontargeting miRNA (pre-NT) or pre-miR-181b-5p mimics, and tissue inhibitor of metalloproteinase 3 (TIMP-3) mRNA (A) and protein (B) were evaluated as described in “Materials and Methods” after 6 days of in vitro decidualization, a time when both TIMP-3 mRNA and protein are significantly elevated. To evaluate if forced expression of miR-181b-5p was associated with an alteration in decidualization, prolactin mRNA (C) and peptide released into the t-HESC conditioned media (D) were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme immunoassay (EIA), respectively, as described in “Materials and Methods.” Data are displayed as the mean ± standard error of the mean (SEM) for all end points from 3 independent observations (n = 3). Data were analyzed by unpaired t test comparing the 2 transfection groups. Significance was set at P < .05 for all comparisons, and P values are displayed within each figure. No significant differences between pre-NT and pre-miR-181b-5p transfection groups were noted in the levels of prolactin mRNA or conditioned media concentrations (P > .05).
Figure 5.
Figure 5.
miR-181b-5p targets the 3′ untranslated region (UTR) of tissue inhibitor of metalloproteinase 3 (TIMP-3) to regulate its expression. A, Sequence alignment of human miR-181b-5p with the 3′ UTR of TIMP-3. Seed sequence of miR-181b-5p that corresponds to the binding sites (3499-3505 and 3573-3580) within the 3′ UTR of TIMP-3 is highlighted in bold and underlined. Mutant constructs incapable of binding miR-181b-5p were generated by deleting both sites 1 and 2 providing a negative control. B, Luciferase assays were conducted as described in “Materials and Methods.” Pre-miR-181b-5p significantly reduced luciferase activity in cells transfected with wild-type 3′ UTR TIMP-3 construct but not in cells transfected with the mutated 3′ UTR of TIMP-3. Pre-miR-NT had no effect on luciferase activity in cells transfected with either the wild-type or mutant 3′ UTR TIMP-3 reporter constructs. Data are displayed as the mean ± standard error of the mean (SEM) and are representative of 4 separate experiments (n = 4). Different letters indicate statistically significant different means within miR between 3′ UTR type while asterisk (*) indicates statistically significant different means within 3′ UTR type between pre-miRs. Data were analyzed using unpaired t tests.
Figure 6.
Figure 6.
CyDye switch, 2-dimensional fluorescence difference gel electrophoresis (2D-DIGE) analysis of pre-NT- and pre-miR-181b-5p-transfected t-HESC proteomes. The t-HESC cells were transfected and protein isolated as described under “Materials and Methods.” Pre-NT-transfected samples were labeled with Cy3 (green) and pre-miR-181b-5p samples with Cy5 (red). Samples were then mixed and separated on analytical 2D-DIGE. The resulting gel was scanned and the merged image is shown where red proteins represent proteins whose expression is higher in the pre-miR-181b-5p-transfected cells and green proteins represent proteins whose expression is higher in the pre-NT-transfected cells. Circled and numbered spots represent proteins that were most differentially expressed. Of these, we determined the identity of 10 proteins that are listed in Table 1 and indicated by the red circles. Molecular weight range is indicated on the vertical axis and isoelectric point range indicated on the horizontal axis. (The color version of this figure is available online.)
Figure 7.
Figure 7.
miR-181b-5p suppresses annexin A2 (Anax2) transcript expression in t-HESC cells. The t-HESC cells were subjected to in vitro decidualization for 2 days with concurrent transfection with either pre-nontargeting (NT) miRNA or pre-miR-181b-5p after which ANXA2 transcript expression (variants 1-4) was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) as described under “Materials and Methods.” Data are displayed as the mean ± standard error of the mean (SEM) for 6 independent observations (n = 6). Data were analyzed by unpaired t test comparing the 2 transfection groups. Significance was set at P < .05 for all comparisons, and P values are displayed within each figure for ANXA2v1 (A), ANXA2v2 (B), and ANXA2v4 (D). No significant differences between pre-NT and pre-miR-181b-5p transfection groups was detected for ANXA2v3 (C; P > .05).
Figure 8.
Figure 8.
miR-181b-5p suppresses annexin A2 (ANXA2) protein expression t-HESC cells under basal conditions. The t-HESC cells were subjected to in vitro decidualization for 2 days with concurrent transfection with either pre-nontargeting (NT) microRNA (miRNA; NT control) or pre-miR-181b-5p after which ANXA2 protein expression was assessed as described under “Materials and Methods” by Western blot analysis. Bar graph represents normalization of ANXA2 to β-actin levels displayed as the mean ± standard error of the mean (SEM) for 3 independent observations (n = 3). Data were analyzed by unpaired t test comparing the 3 transfection groups. Significance was set at P < .05 for all comparisons and asterisk (*) indicates statistical significance between the means. ANXA2 indicates annexin A2; t-HESC, human endometrial stromal cell line; NT, nontargeting.

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