si-SNHG5-FOXF2 inhibits TGF-β1-induced fibrosis in human primary endometrial stromal cells by the Wnt/β-catenin signalling pathway.
si-SNHG5-FOXF2 inhibits TGF-β1-induced fibrosis in human primary endometrial stromal cells by the Wnt/β-catenin signalling pathway.
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DOI：
10.1186/s13287-020-01990-3
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发表时间：
2020-11-11
影响因子：
7.5
通讯作者：
He Y
中科院分区：
文献类型：
--
作者：
Liu L;Chen G;Chen T;Shi W;Hu H;Song K;Huang R;Cai H;He Y
宫腔粘连（IUAs）是子宫内膜纤维化的表现，其特征在于炎症和纤维蛋白原聚集在细胞外基质（ECM）中。现有的IUA治疗干预措施在术后粘连复发和不孕问题的临床环境中效果不足。在这项研究中，我们研究了si-SNHG 5-FOXF 2是否可以作为体外抑制IUA纤维化的分子机制。采用基因芯片技术检测3组正常子宫内膜和6例IUA患者子宫内膜中FOXF 2、TGF-β1和胶原蛋白的表达水平。我们将原代人子宫内膜间质细胞（HESCs）诱导为肌成纤维细胞（MFs），并在不同时间用不同浓度的TGF-β1建立IUA细胞模型。采用染色质免疫沉淀结合全基因组高通量测序（ChIP-seq）技术筛选FOXF 2下游靶基因。我们通过定量逆转录-聚合酶链反应（qRT-PCR）、蛋白质印迹（WB）、免疫组织化学（IHC）、流式细胞术、乙二胺（EdU）和CCK 8测定研究了FOXF 2下调的原代HESC中ECM形成、细胞增殖和Wnt/β-catenin信号通路相关蛋白。我们通过RNA免疫沉淀（RIP）、RNA下拉和荧光原位杂交（FISH）等技术，鉴定出FOXF 2的上游调控基因为长链非编码RNA（lncRNA）SNHG 5。最后，我们研究了FOXF 2表达，ECM形成，细胞增殖和Wnt/β-catenin信号通路相关蛋白在原代HESC后FOXF 2下调。FOXF 2在IUA患者的子宫内膜中高表达。用10 ng/ml TGF-β1处理原代HESC 72 h被认为是开发IUA细胞模型的最有效方法。通过ChIP-seq和ChIP-PCR，FOXF 2调节多个下游靶基因，包括胶原蛋白、波形蛋白（Vim）和细胞周期蛋白D2/DK 4。FOXF 2下调可抑制TGF-β1介导的原发性HESC纤维化，包括ECM形成、细胞增殖和Wnt/β-catenin信号通路相关蛋白表达。我们通过RIP-seq、qRT-PCR、WB和FISH鉴定了lncRNA SNHG 5作为直接调控FOXF 2的上游基因。SNHG 5下调抑制IUA细胞模型中FOXF 2的表达，导致Wnt/β-连环蛋白途径的协同抑制，从而改变离体子宫内膜基质细胞中TGF-β1介导的ECM聚集。si-SNHG 5-FOXF 2对Wnt/β-catenin信号通路和ECM形成的调节有效地抑制了TGF-β1对原代HESC的促纤维化作用。这一发现可为拮抗TGF-β1介导的原代HESCs纤维化提供分子基础。
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. 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. 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. 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.
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DOI：
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期刊：
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影响因子：
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DOI：
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发表时间：
2014-05
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