MiR-204-5p promoted maternally expressed gene 3 (MEG3) through SP1-mediated DNMT1 pathway in trophoblast cells in recurrent miscarriage

Recurrent miscarriage (RM) affects 1-2% of couples. Maternally expressed gene 3 (MEG3) is aberrantly expressed in RM patients. 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. 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. 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. 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.