si-SNHG5-FOXF2 Inhibits TGF-β1-induced Fibrosis in Human Primary Endometrial Stromal Cells by Wnt/β-catenin Signaling 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 si-SNHG5-FOXF2 can serve as a molecular mechanism for inhibition of IUA fibrosis ex vivo.Methods: FOXF2, TGF-β1 and collagens expression levels were measure by a Microarray sequencing analysis in three normal endometrium group and six IUA patients. We induced primary HESCs into MFs to develop an IUA cell model with various concentrations of TGF-β1 at various times. Downstream target genes for 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 signaling 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, EdU and CCK8 assays. We identified lncRNA SNHG5 as the upstream regulatory gene of FOXF2 through RNA immunoprecipitation (RIP), RNA-pulldown and FISH. Finally, we examined FOXF2 expression, ECM formation, cell proliferation and Wnt/ β-catenin signaling pathway-related proteins in primary HESCs with FOXF2 downregulation.Results: FOXF2 was highly expressed in endometrium from patients with IUA. Treatment of primary HESCs with 10ng / 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, 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 signaling pathway-related proteins expressions. We identified lncRNA SNHG5 as an upstream gene that directly regulated FOXF2 by RIP-seq, qRT-PCR, WB and FISH. SNHG5 downregulation suppressed FOXF2 expression in the IUA cell model, resulting in synergistic repression of Wnt/β-catenin signaling pathway activation and thereby altering TGF-β1-mediated ECM aggregation in endometrial stromal cells ex vivo. Conclusions: Regulation of the Wnt/β-catenin signaling pathway and ECM formation by si-SNHG5-FOXF2 effectively inhibited the profibrotic effect of TGF-β1 on primary HESCs. It can provide a molecular basis for antagonizing TGF-β1-mediated fibrosis in primary HESCs.