si-SNHG5-FOXF2 inhibits TGF-ß1-induced fibrosis in human primary endometrial stromal cells by the Wnt/ß-catenin signalling pathway.

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-ß1 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-ß1 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/ß-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/ß-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-ß1 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-ß1-mediated primary HESC fibrosis, including ECM formation, cell proliferation and Wnt/ß-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/ß-catenin pathway, thereby altering TGF-ß1-mediated ECM aggregation in endometrial stromal cells ex vivo. CONCLUSIONS: Regulation of the Wnt/ß-catenin signalling pathway and ECM formation by si-SNHG5-FOXF2 effectively inhibited the profibrotic effect of TGF-ß1 on primary HESCs. This finding can provide a molecular basis for antagonizing TGF-ß1-mediated fibrosis in primary HESCs.

Effects of human adipose-derived mesenchymal stem cells combined with estrogen on regulatory T cells in patients with premature ovarian insufficiency.

OBJECTIVE: To investigate the effects of human adipose-derived mesenchymal stem cells (hADSCs) combined with estrogen on regulatory T cells (Tregs) in patients with premature ovarian insufficiency (POI). METHODS: hADSCs were isolated by enzymatic digestion and identified by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from POI patients and healthy controls. PBMCs were cultured in the following experimental groups: the control group, hADSC group, estrogen group and combined group. The PBMCs in the hADSC group were co-cultured with hADSCs at concentrations of 1×104, 2×104, or 1×105 cells/well, and the estrogen group was co-cultured with 10-9, 10-8, or 10-7mol/L 17ß-estradiol. Cell proliferation was measured with the CCK-8 assay. The percentage of CD4+ CD25+ Foxp3+ Tregs was measured by flow cytometry. The expression levels of Foxp3, TGF-ß1 and IFN-γ were measured by real-time PCR. RESULTS: Treatment with hADSCs, estrogen and their combination promoted Tregs differentiation of PBMCs from POI patients and healthy controls. An increase in the percentage of CD4+ CD25+ Foxp3+ Tregs was observed when PBMCs were co-cultured with hADSCs, 17ß-estradiol and their combination. Foxp3 and TGF-ß1 mRNA expression was higher and IFN-γ mRNA expression was lower in the hADSCs, estrogen and combined groups than in the control group. CONCLUSION: Combined treatment with hADSCs and estrogen played an immunomodulatory role by promoting Tregs proliferation, thereby potentially improving impaired ovarian function.