Abstract
BackgroundMaternally Expressed Gene 3 (MEG3) is expressed at low levels in placental villi during preeclampsia; however, its roles in unexplained recurrent spontaneous abortion (URSA) remain unclear. In this study, we aimed to explore the relationship between MEG3 and URSA.MethodsThe differentially expressed lncRNAs (MEG3) and its downstream genes (RASA1) were identified using bioinformatics analysis of Genomic Spatial Event (GSE) database. The expression levels of MEG3 in embryonic villis (with gestational ages of 49–63 days) and primary trophoblasts were determined using quantitative RT-PCR assay. A mouse model of Embryo implantation, Cell Counting Kit-8 (CCK-8), flow cytometry, and Transwell migration assays were performed to determine the implantation, proliferative, apoptotic, and invasive capacities of trophoblast. The level of phosphorylated core proteins in the RAS-MAPK pathway were analyzed using Western blot assay. The mechanisms of MEG3 in the regulation of RASA1 were studied by RNA pulldown, RNA immunoprecipitation (RIP), DNA pulldown, and chromatin immunoprecipitation (ChIP) assays.ResultsMEG3 had a low expression level in embryonic villis of 102 URSA patients compared with those of 102 normal pregnant women. MEG3 could promote proliferation and invasion, inhibit the apoptosis of primary trophoblast of URSA patients (PT-U cells), as well as promote embryo implantation of mouse. Besides, MEG3 also promoted the phosphorylation of rapidly accelerated fibrosarcoma (Raf), mitogen-activated protein kinase kinase (MEK), and extracellular-signal-regulated kinase (ERK) proteins. The results of RNA pull down and RIP assays showed that MEG3 bound with the enhancer of zeste homolog 2 (EZH2). The DNA pulldown assay revealed that MEG3 could bind to the promoter sequence of the RAS P21 Protein Activator 1 (RASA1) gene. Further, the ChIP assay showed that MEG3 promoted the binding of EZH2 to the promoter region of the RASA1 gene.ConclusionsThe inactivation of MEG3 in embryonic villi association with URSA; MEG3 inhibited the expression of RASA1 by mediating the histone methylation of the promoter of RASA1 gene by EZH2, thereby activating the RAS-MAPK pathway and enhancing the proliferative and invasive capacities of trophoblasts.
Highlights
Recurrent spontaneous abortion (RSA) refers to three or more consecutive spontaneous abortions with multiple complicated etiologies, half of which are unclear and termed as unexplained recurrent spontaneous abortion (URSA)
The inactivation of Maternally Expressed Gene 3 (MEG3) in embryonic villi association with URSA; MEG3 inhibited the expression of RAS P21 Protein Activator 1 (RASA1) by mediating the histone methylation of the promoter of RASA1 gene by enhancer of zeste homolog 2 (EZH2), thereby activating the RASMAPK pathway and enhancing the proliferative and invasive capacities of trophoblasts
MEG3 expression was low in embryonic villous tissues of URSA patients Database analysis of GSE14722 (RNA-seq data of embryonic villi from abortion patients) and GSE9984 (RNAseq data of embryonic villi from normal pregnant women) shows that MEG3 expression was significantly lower in aborted villous tissues than in normal villous tissues (P < 0.01, Fig. 1a)
Summary
Recurrent spontaneous abortion (RSA) refers to three or more consecutive spontaneous abortions with multiple complicated etiologies, half of which are unclear and termed as unexplained recurrent spontaneous abortion (URSA). Studies on lncRNA expression profiles in placental villi from patients with RSA have revealed dysregulated expression of various lncRNAs involved in infections, inflammation, immunity, and apoptosis, thereby suggesting that lncRNAs are closely related to the onset and progression of RSA (Wang 2014; Wang et al 2016). The transcript of maternally expressed gene 3 (MEG3) is an lncRNA that does not encode for protein and has regulatory effect on cell function (Zhang, 2018; Tang et al 2020; Chanda 2018). Expressed Gene 3 (MEG3) is expressed at low levels in placental villi during preeclampsia; its roles in unexplained recurrent spontaneous abortion (URSA) remain unclear. We aimed to explore the relationship between MEG3 and URSA
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