Four and a half LIM domains 1 (FHL1) positively regulates embryo adhesion via binding homeoboxa10 (HOXA10)

This assay aims to study the function of FHL1 in embryo adhesion Endometrial tissue samples were collected from fertile controls (n=14) and patients with recurrent implantation failure (RIF) (n=14). Mice uterus samples of different pregnancy days were obtained. Protein and mRNA were extracted to detect expression levels of FHL1. HEK293T cells, BeWo and Ishikawa cells were used to investigate the molecular mechanism in vitro assay. Detection of FHL1 expression in endometrium of fertile controls,patients with RIF and various pregnant stage mice uterus was performed through western blotting and real-time PCR assay. Association between FHL1 and HOXA10 was confirmed by co-immunoprecipitation, western blotting and luciferase reporter assays. The effect of FHL1-Hoxa10 interaction on embryo adhension was evaluated with BeWo spheroid attachment assay. HomeoboxA10 (HOXA10), a key transcription factor, plays a critical role in embryo adhesion by regulating the expression of downstream target genes, such as β3-integrin (ITGB3). HOXA10-interacting proteins induce embryo adherent effects, and the identification of new HOXA10-binding partners may provide mechanistic insights into the regulation of gene expression during embryo adhesion. We first demonstrated that FHL1 is a HOXA10-associated protein by co-immunoprecipitation assay. FHL1, a member of FHL protein family, whose mRNA and protein levels were revealed to be up-regulated in the mice uterus on Day 4.5 of pregnancy, the window of mice embryo implantation, and overexpression of FHL1 in the Ishikawa cells increased the BeWo spheroids adhesion by 50% (p<0.05). More importantly, we found that FHL1 mRNA and protein in the endometrium from patients with RIF dramatically decreased compared with fertile controls (p<0.05). Secondly, enhanced FHL1 expression in Ishikawa cells promoted endogenous HOXA10 and its target gene ITGB3 expression. Finally, luciferase assay demonstrated that FHL1 overexpression not only improved ITGB3 transcription by 50% (p<0.05), but also promoted HOXA10 mediated ITGB3 transcription by 30% (p<0.05), and short interfering RNA (siRNA)-mediated FHL1 protein knockdown reduced HOXA10 and ITGB3 expression dramatically. While the endogenous Hoxa10 was silenced, the promotion of FHL1 on ITGB3 transcription was undetected. The association between FHL1 and HOXA10 is involved in the regulation of HOXA10-dependent transcription of endometrial genes, which contributes to embryo adhesion.