Abstract
Integrin β3 (ITGB3), which is the target gene of the miRNA let-7 that can be antagonized by long noncoding RNA (lncRNA) H19, is well known to have a critical role in endometrium receptivity. However, the regulation of ITGB3 in cell–cell or cell–extracellular matrix adhesion and invasion for the maintenance of early pregnancy remains unknown. This study aimed to explore the role of the H19/let-7/ITGB3 axis in regulating trophoblastic spheroid adhesion and in vitro invasion ability using the HTR-8/SVneo cell line and to investigate the expression levels of lncRNA H19 and ITGB3 in human products of conception. The in vitro knockdown of H19 resulted in decreased expression of ITGB3 at the mRNA and protein levels and reduced the adhesion and invasion ability. In the embryonic chorion tissue of spontaneous abortion (SA), the expressions of H19 and ITGB3 at both the mRNA and protein levels decreased. The results of quantitative RT-PCR, Western blot analysis, dual-luciferase report gene and functional miRNA let-7 rescue experiments, adhesion assay and in vitro transwell invasion assay confirmed that H19 regulated trophoblastic spheroid adhesion with endometrial stromal cells through the H19/let-7/ITGB3 axis, thereby providing an improved understanding of the molecular mechanism of SA.
Highlights
In all clinically confirmed pregnancies, the incidence of spontaneous abortion (SA) is approximately 15%
HTR-8 cells were transfected with two lentiviral constructs harboring shH19 and PH19 to investigate whether H19 regulated the expression of integrin beta 3 (ITGB3) by competitively binding to miRNA let-7
Combination of ilet-7 and shH19 (Fig. 1I) partially restored the expression of ITGB3 both at the mRNA and protein levels (Fig. 1J and K), and the rescue effect appeared to be significant, when compared with that of combination transfection of the let-7 inhibitor control and shH19. These results suggested that H19 regulated the expression of ITGB3 in the HTR-8 cell line, at least by partial competitive binding to let-7
Summary
In all clinically confirmed pregnancies, the incidence of spontaneous abortion (SA) is approximately 15%. More than 80% of abortions occur within 12 weeks of pregnancy; thereafter, the rate of abortion rapidly declines. Successful human embryo implantation, which requires a viable blastocyst and uterine receptivity, involves a complex and critical multistep process, including embryo apposition/adhesion, followed by penetration and invasion. The initial but fundamental steps of embryo implantation are feto–maternal interaction and cell adhesion and invasion between the blastocyst and endometrial luminal epithelial cells. An in vitro adhesion assay using human endometrial stromal cells (HESCs) and simulated trophoblast spheroid was performed to clarify the adhesion process during embryo implantation. This study investigated the effects of both the downregulation and overexpression of H19 on the adhesion and invasion ability of the HTR-8/SVneo cell line, which was derived from human first-trimester
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