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.
Summary Rice black‐streaked dwarf virus disease (RBSDVD) and southern rice black‐streaked dwarf virus disease (SRBSDVD) are the most destructive viral diseases in rice. Progress is limited in breeding due to lack of resistance resource and inadequate knowledge on the underlying functional gene. Using genome‐wide association study (GWAS), linkage disequilibrium (LD) decay analyses, RNA‐sequencing, and genome editing, we identified a highly RBSDVD‐resistant variety and its first functional gene. A highly RBSDVD‐resistant variety W44 was identified through extensive evaluation of a diverse international rice panel. Seventeen quantitative trait loci (QTLs) were identified among which qRBSDV6‐1 had the largest phenotypic effect. It was finely mapped to a 0.8–1.2 Mb region on chromosome 6, with 62 annotated genes. Analysis of the candidate genes underlying qRBSDV6‐1 showed high expression of aspartic proteinase 47 (OsAP47) in a susceptible variety, W122, and a low resistance variety, W44. OsAP47 overexpressing lines exhibited significantly reduced resistance, while the knockout mutants exhibited significantly reduced SRBSDVD and RBSDVD severity. Furthermore, the resistant allele Hap1 of OsAP47 is almost exclusive to Indica, but rare in Japonica. Results suggest that OsAP47 knockout by editing is effective for improving RBSDVD and SRBSDVD resistance. This study provides genetic information for breeding resistant cultivars.
Phytochelatins (PCs) play important roles in the detoxification of and tolerance to heavy metals in plants. The synthesis of PCs is catalyzed by phytochelatin synthase (PCS), which is activated by heavy metal ions. In this study, we isolated a PCS gene, BnPCS1, from the bast fiber crop ramie (Boehmeria nivea) using the RACE (rapid amplification of cDNA ends) method. The full-length BnPCS1 cDNA is 1,949 bp in length with a 1,518 bp open reading frame (ORF) that encodes a 505 amino acid protein. The deduced BnPCS1 protein has a conserved N-terminus containing the catalytic triad Cys58, His164, Asp182, and a flexible C-terminal region containing a C371C372QETC376VKC379 motif. The BnPCS1 promoter region contains several cis-acting elements involved in phytohormone or abiotic stress responses. Subcellular localization analysis indicates that the BnPCS1-GFP protein localizes to the nucleus and the cytoplasm. Real-time PCR assays show that the expression of BnPCS1 is significantly induced by cadmium (Cd) and the plant hormone abscisic acid (ABA). Overexpression lines of BnPCS1 exhibited better root growth and fresh weight, lower level of MDA and H2O2, and higher Cd accumulation and translocation factor compared to the WT under Cd stress. Taken together, these results could provide new gene resources for phytoremediation of Cd-contaminated soils.
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