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Functional analysis of miRNAs combined with TGF-β1/Smad3 inhibitor in an intrauterine rat adhesion cell model

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Abstract

In this study, we aimed to study the role of miRNAs in intrauterine adhesion (IUA) disease. An IUA cell model was constructed by TGF-β1. Smad3 inhibitor (SIS3) can inhibit the Smad3 signaling pathway and affect the role of TGF-β1; thus, it was used to identify the role of Smad3 and related miRNAs in IUA. Cell number significantly increased in the TGF-β1 group after 72 h and 96 h, respectively, compared with that in the control group (P < 0.05). However, cell proliferation was significantly decreased in the TGF-β1 + SIS3 group (P < 0.0001). Cell apoptosis was increased in the TGF-β1 + SIS3 group compared with that in the TGF-β1 group. Western Blot (WB) analysis suggested that TGF-β1 treatment could effectively increase the expression of α-SMA, COL1, Smad3, and p-Smad3, which could be inhibited by SIS3 treatment. A total of 235 and 530 differentially expressed miRNAs in the TGF-β1 + SIS3 group were significantly up- and downregulated compared with those in the TGF-β1 group, respectively. These differentially expressed miRNAs were enriched in the MAPK and PI3K-AKT pathways. The ten most differentially expressed miRNAs were selected to verify their expressions using quantitative real-time polymerase chain reaction (qPCR). Furthermore, overexpression of rno-miR-3586-3p and rno-miR-455-5p can promote cell proliferation and exacerbate the IUA pathogenic process. However, overexpression of rno-miR-204-3p and rno-miR-3578 can inhibit cell behavior and IUA progression. The above results can provide detailed information for the understanding of IUA molecular mechanisms.

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Data availability

Raw sequencing data presented in this paper are available under the GEO accession number PRJNA598792.

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Funding

This study was supported by the National Natural Science Foundation of China (Grant No. 81701395).

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Authors and Affiliations

  1. Hysteroscopic Centre, Fu Xing Hospital, Capital Medical University, #20 Fuxingmenwai Street, Xicheng District, Beijing, 100038, China

    Shanshan Liu, Xiaowu Huang, Yuhuan Liu, Dongmei Song & Yu Xiao

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  1. Shanshan Liu
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  2. Xiaowu Huang
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  3. Yuhuan Liu
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Contributions

YX conceived and designed the study, and critically revised the manuscript. SL performed the experiments, analyzed the data and drafted the manuscript. XH, YL and DS participated in study design, study implementation and manuscript revision. All authors read and approved the final manuscript.

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Correspondence to Yu Xiao.

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All procedures performed in studies involving animals were in accordance with the ethical standards of the Experimental Animal Welfare Ethics Branch of Biomedical Ethics Committee of Peking University [permit no. LA2018022]. Appropriate guidelines for the use of animals have been followed.

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Liu, S., Huang, X., Liu, Y. et al. Functional analysis of miRNAs combined with TGF-β1/Smad3 inhibitor in an intrauterine rat adhesion cell model. Mol Cell Biochem 470, 15–28 (2020). https://doi.org/10.1007/s11010-020-03741-7

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