PMID- 33176855 OWN - NLM STAT- MEDLINE DCOM- 20210618 LR - 20220531 IS - 1757-6512 (Electronic) IS - 1757-6512 (Linking) VI - 11 IP - 1 DP - 2020 Nov 11 TI - si-SNHG5-FOXF2 inhibits TGF-beta1-induced fibrosis in human primary endometrial stromal cells by the Wnt/beta-catenin signalling pathway. PG - 479 LID - 10.1186/s13287-020-01990-3 [doi] LID - 479 AB - 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-beta1 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-beta1 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/beta-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/beta-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-beta1 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-beta1-mediated primary HESC fibrosis, including ECM formation, cell proliferation and Wnt/beta-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/beta-catenin pathway, thereby altering TGF-beta1-mediated ECM aggregation in endometrial stromal cells ex vivo. CONCLUSIONS: Regulation of the Wnt/beta-catenin signalling pathway and ECM formation by si-SNHG5-FOXF2 effectively inhibited the profibrotic effect of TGF-beta1 on primary HESCs. This finding can provide a molecular basis for antagonizing TGF-beta1-mediated fibrosis in primary HESCs. FAU - Liu, Limin AU - Liu L AD - Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou, China. FAU - Chen, Guobin AU - Chen G AD - Department of Obstetrics and Gynecology, Affiliated Shenzhen Maternity & Child Healthcare Hospital, Southern Medical University, Shenzhen, China. FAU - Chen, Taoliang AU - Chen T AD - The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China. FAU - Shi, Wenjuan AU - Shi W AD - Department of Obstetrics and Gynecology, Affiliated Shenzhen Maternity & Child Healthcare Hospital, Southern Medical University, Shenzhen, China. FAU - Hu, Haiyan AU - Hu H AD - Department of Obstetrics and Gynecology, Affiliated Shenzhen Maternity & Child Healthcare Hospital, Southern Medical University, Shenzhen, China. FAU - Song, Kaijing AU - Song K AD - Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou, China. FAU - Huang, Ruichun AU - Huang R AD - Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou, China. FAU - Cai, Huihua AU - Cai H AD - Department of Obstetrics and Gynecology, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China. chh2004@126.com. FAU - He, Yuanli AU - He Y AD - Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou, China. heyuanli310@163.com. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20201111 PL - England TA - Stem Cell Res Ther JT - Stem cell research & therapy JID - 101527581 RN - 0 (FOXF2 protein, human) RN - 0 (Forkhead Transcription Factors) RN - 0 (RNA, Long Noncoding) RN - 0 (Transforming Growth Factor beta1) RN - 0 (long non-coding RNA SNHG5, human) SB - IM MH - Female MH - Fibrosis MH - Forkhead Transcription Factors/genetics MH - Humans MH - In Situ Hybridization, Fluorescence MH - *RNA, Long Noncoding MH - Stromal Cells MH - *Transforming Growth Factor beta1/genetics MH - *Wnt Signaling Pathway PMC - PMC7656702 OTO - NOTNLM OT - ECM OT - FOXF2 OT - Fibrosis OT - IUA OT - SNHG5 OT - TGF-beta1 COIS- The authors declared that they have no financial or commercial conflicts of interest concerning the research, authorship, or publication of this article. The authors declare no competing interest. EDAT- 2020/11/13 06:00 MHDA- 2021/06/22 06:00 PMCR- 2020/11/11 CRDT- 2020/11/12 05:31 PHST- 2020/08/25 00:00 [received] PHST- 2020/10/21 00:00 [accepted] PHST- 2020/11/12 05:31 [entrez] PHST- 2020/11/13 06:00 [pubmed] PHST- 2021/06/22 06:00 [medline] PHST- 2020/11/11 00:00 [pmc-release] AID - 10.1186/s13287-020-01990-3 [pii] AID - 1990 [pii] AID - 10.1186/s13287-020-01990-3 [doi] PST - epublish SO - Stem Cell Res Ther. 2020 Nov 11;11(1):479. doi: 10.1186/s13287-020-01990-3.