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Updated: May 15, 2025
Derivation of Mouse Trophoblast Stem Cells from Blastocysts
Published on: June 8, 2010
MiR-204-5p promoted maternally expressed gene 3 (MEG3) through SP1-mediated DNMT1 pathway in trophoblast cells in
Shasha Liu1, Yue Wang1, Yuling Guo1
1Department of Obstetrics, Tianjin Central Hospital of Obstetrics and Gynecology, China.
Background:
Recurrent miscarriage (RM) affects 1-2% of couples. Maternally expressed gene 3 (MEG3) is aberrantly expressed in RM patients.
Objectives:
To investigate a novel regulatory mechanism, we examined the miR-204-5p/Specificity protein 1 (SP1)/DNA methyltransferase 1 (DNMT1)/MEG3 axis in the trophoblast cell line HTR-8/SVneo.
Material And Methods:
Human trophoblast cell line HTR-8/SVneo was used and cells were transfected with siRNA targeting SP1, miR-204-5p mimics, pcDNA3.1-DNMT1, or their negative controls (NCs). The methylation inhibitor, 5-azadC, was used to treat the cells transfected with pcDNA3.1-SP1. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) method was used to examine the relative RNA levels of SP1, DNMT1 and MEG3. Western blot assay was performed to measure the protein levels of SP1 and DNMT1. The dual-luciferase reporter gene assay was used to validate the miR-204-5p bindings to SP1. Functional assays were utilized to assess cell apoptosis, colony formation, migration, and invasion.
Results:
SP1 knockdown inhibited DNMT1 and increased MEG3 expression. The expression of MEG3 was enhanced by methylation inhibition through 5-azadC, but SP1 upregulation reversed this effect. SP1 knockdown increased apoptosis and decreased migration and invasion, which was reversed by DNMT1 overexpression. SP1 was targeted and inhibited by miR-204-5p. miR-204-5p also inhibited DNMT1, and enhanced the expression of MEG3. miR-204-5p inhibited cell proliferation, migration and invasion, and promoted apoptosis. Overexpression of SP1 partially reversed these effects by activating DNMT1 and inhibiting MEG3.
Conclusions:
miR-204-5p promoted MEG3 expression in trophoblast cells via SP1-mediated DNMT1 inhibition, leading to reduced cell migration, proliferation and invasion, as well as increased apoptosis. This study reveals a novel regulatory axis in trophoblast cells, providing insights into potential regulatory mechanisms in RM.
Insights
A novel regulatory axis involving miR-204-5p, SP1, DNMT1, and MEG3 was identified in trophoblast cells. This pathway influences cell behavior and offers insights into recurrent miscarriage mechanisms.
Area of Science:
- Reproductive Biology
- Molecular Genetics
- Cell Biology
Background:
- Recurrent miscarriage (RM) impacts 1-2% of couples.
- Aberrant expression of Maternally expressed gene 3 (MEG3) is observed in RM patients.
Purpose of the Study:
- To investigate the miR-204-5p/Specificity protein 1 (SP1)/DNA methyltransferase 1 (DNMT1)/MEG3 regulatory axis.
- To elucidate the role of this axis in trophoblast cell function.
Main Methods:
- Utilized the human trophoblast cell line HTR-8/SVneo.
- Employed siRNA, mimics, gene overexpression, and methylation inhibition (5-azadC).
- Assessed gene and protein expression via RT-qPCR and Western blot; validated interactions using dual-luciferase reporter assay; evaluated cell functions including apoptosis, proliferation, migration, and invasion.
Main Results:
- SP1 knockdown inhibited DNMT1 and increased MEG3 expression.
- miR-204-5p targeted SP1, inhibiting DNMT1 and enhancing MEG3 expression.
- This axis modulated trophoblast cell apoptosis, proliferation, migration, and invasion.
Conclusions:
- miR-204-5p promotes MEG3 expression in trophoblast cells by inhibiting SP1-mediated DNMT1.
- This pathway reduces cell migration, proliferation, and invasion while increasing apoptosis.
- Identified a novel regulatory axis providing potential insights into recurrent miscarriage.
