miR-338-5p Targets Epidermal Growth Factor-Containing Fibulin-Like Extracellular Matrix Protein 1 to Inhibit the Growth and Invasion of Trophoblast Cells in Selective Intrauterine Growth Restriction

Abstract

Selective intrauterine growth restriction (sIUGR) is a disorder of monochorionic (MC) twin pregnancies. However, the underlying mechanism remains largely unknown. Trophoblast cells are the major component of the placenta. Dysfunction of trophoblast cells is associated with placental dysfunction. Our previous study identified miR-338-5p is downregulated in placenta tissues sharing larger twins of sIUGR. In the present study, we aimed to investigate the role of miR-338-5p in trophoblast cells and explored its target. Our results further indicated that miR-338-5p was downregulated in placental tissues supporting larger twins of sIUGR, whereas epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) was upregulated. Moreover, miR-338-5p overexpression suppressed the growth and invasion of trophoblast cells. Importantly, results from luciferase reporter assay demonstrated that miR-338-5p bound on the 3'-UTR of EFEMP1. miR-338-5p suppressed the growth and invasion of trophoblast cells via targeting EFEMP1. Further, miR-338-5p/EFEMP1 might disrupt the function of trophoblast cells via inhibiting the phosphorylation of AKT.

Keywords: AKT; EFEMP1; Trophoblast cell; miR-338-5p; sIUGR.

Fig. 1

Differential expression of miR-338-5p and EFEMP1 in sIUGR twins. a miR-338-5p was upregulated in the placental share of the smaller fetus in sIUGR pregnancies, *** p < 0.001 vs high body weight. b The relative protein content of EFEMP1 was downregulated in the placental share of the smaller fetus in sIUGR pregnancies, ** p < 0.01 vs high body weight. NH: high body weight in normal twin, NL: low body weight in normal twin, sH: high body weight in sIUGR twin, sL: low body weight in sIUGR twin

Fig. 2

miR-338-5p overexpression suppressed the growth and invasion of trophoblast cells. a The level of miR-338-5p was significantly upregulated in mimic transfected cells, *** p < 0.01 vs miNC. b miR-338-5p overexpression inhibited the proliferation of trophoblast cells, * p < 0.05 vs miNC. c miR-338-5p mimic promoted the apoptosis of trophoblast cells, *** p < 0.001 vs miNC. d miR-338-5p overexpression suppressed the invasion of trophoblast cells, *** p < 0.001 vs miNC. e The protein contents of cleaved caspase-3, EFEMP1, and p-AKT/AKT were examined in blank, miNC, and mimic cells, *** p < 0.001 vs miNC

Fig. 3

miR-338-5p targeted EFEMP1 in human embryonic kidney cells through binding on its 3′-UTR. a The putative miR-338-5p binding site in the 3′-untranslated region (3′-UTR) of EFEMP1 and the corresponding mutant sequence are presented as indicated. b The luciferase activity of the reporter driven by the wild type EFEMP1 3′-UTR was deeply suppressed in mimic cells as indicated above, *** p < 0.001 vs miNC. c and d The relative mRNA and protein levels of EFEMP1 were significantly upregulated in oeEFEMP1 transfected cells, *** p < 0.001 vs oeNC

Fig. 4

EFEMP1 overexpression promoted the growth and invasion of trophoblast cells. a The level of miR-338-5p was examined in different cells as indicated, *** p < 0.001 vs miNC. b EFEMP1 overexpression promoted the proliferation of trophoblast cells, ** p < 0.001 vs miNC+oeNC,! p < 0.05 vs miNC + oeEFEMP1,!!! p < 0.001 vs miNC + oeEFEMP1. c EFEMP1 overexpression inhibited the apoptosis of trophoblast cells; ** p < 0.01 vs miNC+oeNC; *** p < 0.001 vs miNC+oeNC;! p < 0.05 vs miNC+oeEFEMP1;!!! p < 0.001 vs miNC+oeEFEMP1; ### p < 0.001 vs mimic+oeEFEMP1. d EFEMP1 overexpression promoted the invasion of trophoblast cells; *** p < 0.001 vs miNC+oeNC;!!! p < 0.001 vs miNC + oeEFEMP1; ### p < 0.001 vs mimic + oeEFEMP1. e The relative protein contents of cleaved caspase-3, EFEMP1, and p-AKT/AKT were examined in different cells as indicated, * p < 0.05 vs miNC+oeNC, *** p < 0.001 vs miNC+oeNC,!!! p < 0.001 vs miNC + oeEFEMP1