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MicroRNA-145 targets Smad1 in endometrial stromal cells and regulates decidualization in rat

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Abstract

Decidualization of endometrial stromal cells is the pre-requisite for the embryo implantation and establishment of pregnancy. Although known to be regulated by several factors, the process of regulation of decidualization by miRNAs is largely unknown. Previous reports suggest that the upregulated expression of miR-145 is associated with repeated implantation failure. The current study was aimed to identify and validate the role of miR-145 in regulating stromal cell decidualization and the mechanism involved therein. Expression of miR-145 was found to be downregulated during the decidualization period of early pregnancy and also in artificially induced decidualization in rat uterus. During in vitro decidualization in rat endometrial stromal cells (ESCs), the overexpression of mimic miR-145 attenuated the progression of decidualization. Biochemical marker alkaline phosphatase and protein markers (insulin-like growth factor binding protein, cyclin D3) were also suppressed in miR-145 mimic-transfected cells as compared to normal decidualized cells. Bioinformatic analysis and luciferase reporter assay confirmed that Smad1 is the direct target of miR-145. Differentiation of ESCs was inhibited in miR-145 mimic-transfected cells which occurred via downregulating the target Smad1 along with its downstream p-Smad1/5/8 and Wnt-4. Pre-treatment of ESCs with Smad1 siRNA resulted in downregulated expression of p-Smad1/5/8, Wnt-4, Cox-2, and VEGF. In addition, miR-145 overexpression resulted in the loss of angiogenic factors Cox-2, MMP-9, and VEGF, indicating suppression of the process of angiogenesis. Migration of human umbilical vein endothelial cells was also attenuated in the presence of conditioned media obtained from miR-145-transfected decidualizing cells. In conclusion, the study demonstrated the role of miR-145 in regulation of progression of decidualization which is mediated through inhibition of Smad1.

Key messages

  • MiR-145 expression is downregulated during decidualization in the rat uterus.

  • Overexpression of miR-145 inhibited the decidualization progression.

  • MiR-145 suppressed the migration and invasion of HUVECs.

  • MiR-145 downregulated Smad1 which suppresses Smad1/5/8, Wnt-4, MMP-9, Cox-2, and VEGF.

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Abbreviations

miRNA:

Micro RNA

ESCs:

Endometrial stromal cells

PBS:

Phosphate-buffered saline

UTR:

Untranslated region

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Acknowledgments

We are thankful to Dr. PK Agnihotri for help in histology work; Mr. AL Vishwakarma, Sophisticated Analytical Instrument Facility, for help in flow cytometry experiments; and Dr. Mukesh Srivastava, Biometry & Statistics Division, CSIR-CDRI, for his help in statistical analysis. Financial support was provided by the Council of Scientific & Industrial Research, India. One of the authors (V.K.S.) is a recipient of a senior research fellowship from the Council of Scientific and Industrial Research, India. This is CDRI communication number 9793.

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Authors and Affiliations

  1. Division of Endocrinology, CSIR-Central Drug Research Institute|, Lucknow, Uttar Pradesh, 226031, India

    Vijay K. Sirohi, Kanchan Gupta, Radhika Kapoor & Anila Dwivedi

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  1. Vijay K. Sirohi
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Correspondence to Anila Dwivedi.

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All the experimental protocols were followed as per CPCSEA guidelines and approved by the Institutional Animal Ethics Committee (IAEC), CSIR- Central Drug Research Institute, Lucknow.

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Fig. S1

(A) Graph showing the wet weight of uteri collected from rats with artificially induced decidualization. Results are expressed as mean ± SE, p values are **p < 0.01 vs. control, n = 5 (B) Representative images of hematoxylin- and eosin- stained cross sections of uterine horns from un-stimulated and oil-stimulated rats. Images were captured by light microscope at 10x and 40x magnifications. (C) Representative images of immunofluorescence of cytokeratin and vimentin in primary rat ESCs. (JPG 1263 kb)

Fig. S2

Effect of miR-145 mimic on cell death of endometrial stromal cells (AnnexinV/PI staining). Briefly, cells were probed with fluorescein isothiocynate (FITC)-conjugated Annexin-V and PI for 15 min in dark. The staining profiles were determined with FACScan (BD Biosciences, CA, USA) and Cell-Quest software. Representative images of quadrants showing live (AV−-/PI−-), apoptotic (AV+/PI−- and AV+/PI+) and necrotic (AV−-/PI+) endometrial stromal cells undergoing decidualization pretreated with either mimic control or miR-145 mimic (upper panel).The graph in lower panel shows the percent live, apoptotic and necrotic cells in groups indicated. Values are mean ± SEM, n = 3. (JPG 723 kb)

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Sirohi, V.K., Gupta, K., Kapoor, R. et al. MicroRNA-145 targets Smad1 in endometrial stromal cells and regulates decidualization in rat. J Mol Med 97, 509–522 (2019). https://doi.org/10.1007/s00109-019-01744-6

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  • DOI: https://doi.org/10.1007/s00109-019-01744-6

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