si-SNHG5-FOXF2 inhibits TGF-β1-induced fibrosis in human primary endometrial stromal cells by the Wnt/β-catenin signalling pathway

Abstract

Background Intrauterine adhesions (IUAs) are manifestations of endometrial fibrosis characterized by inflammation and fibrinogen aggregation in the extracellular matrix (ECM). The available therapeutic interventions for IUA are insufficiently effective in the clinical setting for postoperative adhesion recurrence and infertility problems. In this study, we investigated whether si-SNHG5-FOXF2 can serve as a molecular mechanism for the inhibition of IUA fibrosis ex vivo. Methods FOXF2, TGF-β1 and collagen expression levels were measured by microarray sequencing analysis in three normal endometrium groups and six IUA patients. We induced primary human endometrial stromal cells (HESCs) into myofibroblasts (MFs) to develop an IUA cell model with various concentrations of TGF-β1 at various times. Downstream target genes of FOXF2 were screened by chromatin immunoprecipitation combined with whole-genome high-throughput sequencing (ChIP-seq). We investigated ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related proteins in primary HESCs with FOXF2 downregulation by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting (WB), immunohistochemistry (IHC), flow cytometry, ethylenediurea (EdU) and CCK8 assays. We identified long noncoding RNAs (lncRNA) SNHG5 as the upstream regulatory gene of FOXF2 through RNA immunoprecipitation (RIP), RNA pulldown and fluorescence in situ hybridization (FISH). Finally, we examined FOXF2 expression, ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related proteins in primary HESCs upon FOXF2 downregulation. Results FOXF2 was highly expressed in the endometrium of patients with IUA. Treatment of primary HESCs with 10 ng/ml TGF-β1 for 72 h was found to be most effective for developing an IUA cell model. FOXF2 regulated multiple downstream target genes, including collagen, vimentin (VIM) and cyclin D2/DK4, by ChIP-seq and ChIP-PCR. FOXF2 downregulation inhibited TGF-β1-mediated primary HESC fibrosis, including ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related protein expression. We identified lncRNA SNHG5 as an upstream gene that directly regulates FOXF2 by RIP-seq, qRT-PCR, WB and FISH. SNHG5 downregulation suppressed FOXF2 expression in the IUA cell model, resulting in synergistic repression of the Wnt/β-catenin pathway, thereby altering TGF-β1-mediated ECM aggregation in endometrial stromal cells ex vivo. Conclusions Regulation of the Wnt/β-catenin signalling pathway and ECM formation by si-SNHG5-FOXF2 effectively inhibited the profibrotic effect of TGF-β1 on primary HESCs. This finding can provide a molecular basis for antagonizing TGF-β1-mediated fibrosis in primary HESCs.

Role of noncoding RNA in the pathophysiology and treatment of intrauterine adhesion

“…Whether other cytokines are involved in the regulation has not been further studied. Another study showed that lncRNA-SNHG5 regulates Wnt/β-catenin by targeting FOXF2 and participates in fibrosis ( Liu et al, 2020c ). This experiment verified that lncRNA-SNHG5 is highly expressed in the IUA cell model and positively regulates FOXF2, which verified that silencing lncRNA-SNHG5 could effectively block the Wnt/β-catenin pathway and inhibit the process of fibrosis.…”

Transient and Prolonged Activation of Wnt Signaling Contribute Oppositely to the Pathogenesis of Asherman’s Syndrome

“…Previously, Wnt signaling has been reported to contribute to stem cell aging and increase tissue fibrosis [ 25 ]. Other reports have also indicated the role of Wnt signaling in the ECM formation in the endometrium [ 26 ], which may also contribute to fibrosis formation. Thus, it is possible that the pathogenesis of IUA is partially due to hyper-activation of Wnt signaling, which leads to increased growth of the fibrotic tissue.…”

Therapeutic Role of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Female Reproductive Diseases

“…Moreover, it has been reported that the aberrantly activated Wnt/β-catenin pathway was confirmed to stimulate TGF-β-mediated fibrosis and mediate fibrogenesis in the endometrium ( 65 , 66 ). FOXF2 protein, which activated Wnt/β-catenin pathway and upregulated Collagen Type V Alpha 2 (COL5A2) transcription in the endometrium, was reported to promote fibrogenesis in the IUA as well ( 67 , 68 ). Therefore, the interaction between many proteins or signaling pathways is of significance to the pathogenesis of IUA.…”

“…Interestingly, these signaling pathways not only were functional in promoting angiogenesis, but also exerted therapeutic effects via other mechanisms. For instance, Wnt/β-catenin pathway promoted angiogenesis, and was involved in TGF-β1-mediated fibrosis ( 67 , 142 ). Therefore, further researches need to characterize the role of pathways in ameliorating IUA by MSC-EVs.…”