MicroRNA-145 targets Smad1 in endometrial stromal cells and regulates decidualization in rat
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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.
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.
KeywordsMiRNA-145 Smad1 Decidualization
Endometrial stromal cells
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.
Compliance with ethical standards
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.
Conflict of interest
The authors declare that they have no conflict of interest.
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