Friend leukemia integration 1 overexpression decreases endometrial receptivity and induces embryo implantation failure by promoting PART1 transcription in the endometrial epithelial cells

Abstract

Background: In vitro fertilization-embryo transfer (IVF-ET) is a crucial assisted reproductive technology for treating infertility. However, recurrent implantation failure (RIF), a significant challenge in IVF-ET success, remains unresolved. This study aimed to explore the role and mechanism of FLI1 in endometrial receptivity and RIF.

Methods: Differential endometrial cell proportions between patients with RIF and control subjects were assessed using single-cell RNA sequencing (scRNA-seq) analysis. The chromatin accessibility of FLI1 in the luteal endometrial tissue of patients with RIF and control subjects was examined using the single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq). FLI1 mRNA and protein levels were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Cell viability and migration were examined via cell counting kit (CCK)-8 and scratch healing assays. Epithelial-mesenchymal transition markers were analyzed using western blotting. Mechanisms underlying FLI1's regulation of PART1 transcription and expression in endometrial epithelial cells were explored using chromatin immunoprecipitation and dual-luciferase reporter assays. Adeno-associated virus (AAV) carrying epithelial cell-specific FLI1/PART1 overexpression sequences was uterinely injected in mice to assess FLI1/PART1 effects.

Results: scRNA-seq revealed diminished endometrial epithelial cell proportions in RIF patients. Meanwhile, scATAC-seq indicated enhanced chromatin accessibility of FLI1 in these cells. FLI1 exhibited specific expression in RIF patients' endometrial epithelial cells. Specific FLI1 overexpression inhibited embryo implantation, while knockdown enhanced it. Pregnant mice injected with AAV encoding FLI1 overexpression had significantly lower implantation than AAV-negative controls. FLI1 binding to PART1 promoter heightened PART1 transcription and expression in endometrial epithelial cells. Rescue experiments illustrated FLI1's role in embryo implantation by boosting PART1 expression. PART1 was notably elevated in RIF patients' luteal endometrial tissue and non-receptive endometrial epithelial cells (HEC-1-A). Specific PART1 overexpression dampened embryo implantation, whereas knockdown promoted it. Pregnant mice injected with AAV encoding PART1 had lower implantation than negative controls. PART1 knockdown mitigated FLI1's inhibitory impact on HEC-1-A cell viability and migration.

Conclusions: FLI1 overexpression in the endometrial epithelial cells of patients with RIF inhibited embryo implantation by binding to the PART1 promoter region to promote PART1 expression. These findings can aid in the development of novel therapeutic targets for RIF.

Keywords: Endometrial receptivity; FLI1; PART1; Recurrent implantation failure (RIF); Single cell chromatin accessibility sequencing (scATAC-seq).

Figure 1. Single-cell clustering analysis and identification of cells in the endometrial tissue.

(A) Single-cell t-SNE clustering and identification of cells in the endometrial tissue. (B) Single-cell t-SNE clustering and identification of cells in the endometrial tissue of the RIF (left) and control (right) groups; (C) Heatmap showing the relative expression levels of the top 10 genes in each cell group. Visualization of key genes in each cell group (D–K). t-SNE, t-distributed stochastic neighbor embedding; RIF, recurrent implantation failure; NC, control group.

Figure 2. Decreased endometrial receptivity in patients with RIF is associated with downregulated endometrial epithelial cell content and aberrant FLI1 expression.

(A) The number and proportion of eight cell populations in the endometrial tissue of the RIF and control groups. (B) The scATAC-seq data were used to analyze the peaks of FLI1. The height of each color peak represents the opening degree of corresponding cell type chromatin; (C) The mRNA expression of FLI1 in the endometrial epithelial cells was examined using qRT-PCR analysis. (D) The protein expression of FLI1 in endometrial epithelial cells was examined using western blotting. scATAC, single-cell assay for transposase accessible chromatin with high-throughput sequencing; NC, control group; qRT-PCR, quantitative real-time polymerase chain reaction; RIF, recurrent implantation failure; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3. Effect of FLI1 on cell functions.

(A) The FLI1 expression levels in HEC-1-A, Ishikawa, and RL95-2 cells were examined using qRT-PCR analysis. Verification of the efficiency of FLI1 overexpression and silencing plasmid stable transfection using qRT-PCR (B) and western blotting (C) in HEC-1-A and Ishikawa cells. (D) The proliferation of HEC-1-A and Ishikawa cells transfected with OE-NC and OE-FLI1 plasmids was examined using the CCK-8 assay. The invasive ability of HEC-1-A (E) and Ishikawa (F) cells transfected with OE-NC and OE-FLI1 plasmids was analyzed using the scratch test. The protein expression of vimentin and E-cadherin in HEC-1-A and Ishikawa cells transfected with OE-NC and OE-FLI1 plasmids was examined using western blotting (G–H). (I) The effect of FLI1 overexpression on the ability of Ishikawa cells to adhere to BeWo cells and the effect of FLI1 knockdown on the ability of HEC-1-A cells to adhere to BeWo cells. OE-FLI1 and OE-NC, FLI1 overexpression plasmid and its negative control plasmid, respectively; sh-FLI1 and sh-NC, FLI1 knockdown plasmid and its negative control plasmid; NC, control group; qRT-PCR, quantitative real-time polymerase chain reaction; CCK-8, cell counting kit-8; OE, overexpression. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4. Epithelial cell-specific FLI1 overexpression inhibits mouse embryo implantation.

The expression levels of FLI1 in the mouse endometrial epithelial cells were analyzed using qRT-PCR and western blotting (A and B). (C) The effect of epithelial cell-specific FLI1 overexpression on the number of implanted embryos in pregnant mice. AVV-OE-FLI1 and AVV-OE-NC, adeno-associated virus vector encoding FLI1 overexpression plasmid and its negative control plasmid, respectively. qRT-PCR, quantitative real-time polymerase chain reaction. **p < 0.01; ***p < 0.001.

Figure 5. LOC100505912 and PART1 are upregulated in the endometrial tissue of patients.

(A) Heatmap showing the differentially expressed long non-coding RNAs (lncRNAs) in the GSE26787 dataset. (B) Heatmap showing the differentially expressed lncRNAs in the GSE111974 dataset. (C) The Venn plot and bar plot showing the common differentially expressed lncRNAs between the GSE26787 and GSE111974 datasets. Ctrl and RIF, endometrial tissues of healthy pregnant women and patients with recurrent implantation failure, respectively; DElncRNAs: differentially expressed lncRNAs. ** p < 0.01.

Figure 6. FLI1 promotes the transcription of PART1 by binding to its promoter.

qRT-PCR analysis of the effect of FLI1 overexpression or knockdown on the PART1 and LOC100505912 levels in endometrial epithelial cells (A–B). (C) The binding site of FLI1 in the PART1 promoter was predicted using JASPAR. (D) The interaction between FLI1 and PART1 promoter was examined using the ChIP assay. (E) The results of the dual-luciferase reporter assay demonstrated that FLI1 regulates PART1 transcription by binding to its promoter. sh-FLI1 and sh-NC, FLI1 knockdown plasmid and its negative control plasmid, respectively; OE-FLI1 and OE-NC, FLI1 overexpression plasmid and its negative control plasmid, respectively; qRT-PCR, quantitative real-time polymerase chain reaction; ChIP, chromatin immunoprecipitation. **p < 0.01; ***p < 0.001.

Figure 7. Effects of epithelial cell-specific PART1 overexpression on mouse embryo implantation and endometrial epithelial cell function.

(A) The mRNA expression level of PART1 in the mouse endometrial epithelial cells and the effect of PART1 overexpression on the number of implanted embryos in pregnant mice. (B) The expression level of PART1 in the endometrial epithelial cells and the analysis of endometrial epithelial cell proliferation in different groups using the CCK-8 assay. (C) Cell invasion ability was examined using the scratch test. (D) Western blotting analysis of vimentin and E-cadherin in Ishikawa cells; (E) Cell adhesion ability was examined using the BeWo cells. AVV-OE PART1 and AVV-OE-NC, adeno-associated virus encoding PART1 overexpression construct and its negative control construct, respectively; OE-FLI1 and OE-NC, FLI1 overexpression plasmid and its negative control plasmid, respectively; sh-PART1 and sh-NC, PART1 knockdown plasmid and its negative control plasmid, respectively; CCK-8, cell counting kit-8; **p < 0.01; ***p < 0.001.