miR-18a Contributes to Preeclampsia by Downregulating Smad2 (Full Length) and Reducing TGF-β Signaling

Abstract

The study investigated the regulation of Smad2 by miR-18a and its role in preeclampsia (PE). Bioinformatics analysis showed that both Smad2 and Smad3 were the predicted targets for miR-18a. Mass spectrum analysis showed that two mature Smad2 isoforms existed in human placenta: full length, Smad2(FL), and that lacking exon3, Smad2(Δexon3). The protein level of Smad2(FL), but not Smad2(Δexon3) or Smad3, was significantly increased in severe PE (sPE) placenta, which was inversely correlated with the level of miR-18a. Elevated Smad2(FL) phosphorylation level appeared in sPE placenta, and Smad2 was colocalized with miR-18a in various subtypes of trophoblasts in human placenta. Smad2(FL) was validated as the direct target of miR-18a in HTR8/SVneo cells. miR-18a enhanced trophoblast cell invasion, which was blocked by the overexpression of Smad2(FL). Furthermore, overexpression of miR-18a repressed Smad2 activation and the inhibition of trophoblast cell invasion by transforming growth factor-β (TGF-β). In conclusion, our results suggest that miR-18a inhibits the expression of Smad2(FL), but not Smad2(Δexon3) or Smad3, which can reduce TGF-β signaling, leading to the enhancement of trophoblast cell invasion. A lack of miR-18a, which results in the upregulation of Smad2(FL), contributes to the development of PE.

Keywords: Smad2; cell invasion; miR-18a; placental trophoblast; preeclampsia.

Graphical abstract

Figure 1

Differential Expression Patterns of pri-miR-18a and Mature miR-18a in sPE Placentas (A–D) Quantitative real-time PCR experiments were performed to measure the expression levels of pri-miR-18a (A and B) and mature miR-18a (C and D) in the chorionic plates (A and C) and basal plates (B and D) of the placentas derived from sPE patients (n = 13) and normal pregnant women (n = 32). Data are presented as mean ± SD. ∗p < 0.05. (E) miR-18a expression levels change across gestation. At least 3 samples were collected and analyzed in each gestational week. Data are presented as mean ± SD. ∗Compared to the miR-18a level at gestational weeks 6–7 (6–7w), p < 0.05.

Figure 2

Differential Expression Patterns of Smad3 in Placentas Derived from sPE Patients and Normal Pregnant Women (A–D) Quantitative real-time PCR (A and B) and western blotting (C and D) were performed to measure the expression of Smad3 in chorionic (A and C) and basal plates (B and D) of placentas derived from sPE patients (n = 13) and normal pregnant women (n = 32). Data are presented as mean ± SD.

Figure 3

Differential Expression Patterns of Smad2 Isoforms and p-Smad2 Isoforms in Placentas Derived from sPE Patients and Normal Pregnant Women (A and B) Quantitative real-time PCR were performed to measure the expression of the Smad2(FL) and Smad2(Δexon3) in chorionic (A) and basal plates (B) of placentas derived from sPE patients (n = 13) and normal pregnant women (n = 32). (C–F) Western blotting was performed to measure the expression of Smad2(FL), Smad2(Δexon3), p-Smad2(FL), and p-Smad2(Δexon3) in chorionic (C and E) and basal plates (D and F) of placentas derived from sPE patients (n = 8) and normal pregnant women (n = 8). (G–J) The inverse correlation between the miR-18a and Smad2(FL) (or p-Smad2(FL)) expression in the placental chorionic (G and I) and basal (H and J) plates of the studied individuals was shown. Data are presented as mean ± SD. ∗p < 0.05.

Figure 4

Colocalization of miR-18a and Smad2 in Human Placenta (A–D) In situ hybridization and immunochemistry experiments showing the colocalization of miR-18a (A and B) and Smad2 (C and D) in human normal placentas at gestational weeks 7–8. (E and F) Immunostaining for CK-8 was used to mark the interstitial trophoblasts in placentas using a section adjacent to that used in (A)–(D). STB, syncytiotrophoblasts; CTB, cytotrophoblast cells; iEVT, interstitial extravillous trophoblast cells. Scale bars, 20 μm.

Figure 5

Validation of Smad2(FL) as the Direct Target of miR-18a in Human Trophoblast Cells (A) Quantitative real-time PCR to reveal change of the Smad2(FL) mRNA level in HTR8/ SVneo cells transfected with negative control (NC), miR-18a mimics, or Smad2 siRNA. (B) Western blot analysis to show change of Smad2(FL) protein level in HTR8/SVneo cells transfected with NC, miR-18a, or si-Smad2. (C) Schematic representation of luciferase constructs used for reporter assays. The construct containing the region complementary to the seed sequence for miR-18a in the 3′ untranslated region (UTR) segment of the human Smad2(FL) gene is shown as BD-WT, and mutant construct is shown as BD-MUT (mutation sites). (D) Luciferase activity measured in HTR8/SVneo cells transfected with BD-WT or BD-MUT luciferase constructs together with miR-18a or NC. All experiments were performed with triplicate in at least 3 independent experiments. Data above are presented as mean ± SD. ∗p < 0.05.

Figure 6

Effects of miR-18a on Cell Invasion and Proliferation in HTR8/SVneo Cells (A and B) Quantitative real-time PCR results to reveal the miR-18a expression in HTR8/SVneo cells transfected with miR-18a mimics and scramble control (NC) (A) and miR-18a inhibitor or scramble inhibitor (inhibitor-NC) (B). The level of miR-18a was adjusted by the value of U6, and the relative value is presented as the mean ± SD, based on 3 independent experiments. ∗p < 0.05. (C and D) Transwell insert assay to examine cell invasiveness in HTR8/SVneo cells transfected with NC and miR-18a (C) and inhibitor NC or miR-18a inhibitor (D). The invasion index is presented as mean ± SD, based on 3 independent experiments. ∗p < 0.05. (E) The effect of miR-18a on the cell growth of HTR8/SVneo cells. The data are presented as the mean ± SD, according to the results of three independently repeated experiments. (F) The effect of miR-18a on the cell cycle of HTR8/SVneo cells. The data are presented as the mean ± SD, according to the results of three independently repeated experiments. (G) Smad2(FL) rescued the effect of miR-18a on cell invasion in HTR8/SVneo cells. The cells were transfected with miR-18a and Smad2 alone or in combination with scramble siRNA (NC) or pcDNA4 vector (pcDNA4) as corresponding NC. The Transwell insert assay was performed to monitor cell invasiveness. The bar chart represents statistical analysis based on 3 independently repeated experiments. Data are presented as mean ± SD. ∗p < 0.05.

Figure 7

miR-18a Blocks TGF-β Signaling in HTR8/SVneo Cells (A–C) miR-18a inhibited Smad2(FL) expression and activation induced by TGF-β1. (D) miR-18a reversed the inhibitory effect of TGF-β1 on cell invasion. All experiments were carried out with triplicate in at least three independent experiments. The data above were presented as mean ± SD. ∗p < 0.05.

Figure 8

Proposed Roles of miR-18a in Placenta Development and Preeclampsia In preeclamptic placenta, an increase in TGF-β expression and a decrease in miR-18a expression will cause overactivation of Smad2(FL), leading to insufficient invasion of trophoblast cells.