Dataset Information

The long noncoding RNA MEG3 regulates Ras-MAPK pathway through RASA1 in trophoblast and is associated with unexplained recurrent spontaneous abortion.

ABSTRACT:

Background

Maternally 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.

Methods

The 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.

Results

MEG3 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.

Conclusions

The 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.

SUBMITTER: Zhang J

PROVIDER: S-EPMC8265043 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundUnexplained recurrent spontaneous abortion (URSA) is a common pregnancy complication and the etiology is unknown. URSA-associated lncRNAs are expected to be potential biomarkers for diagnosis, and might be related to the disease pathogenesis.ObjectiveTo investigate differential lncRNAs in peripheral blood of non-pregnant URSA patients and matched healthy control women and to explore the possible mechanism of differential lncRNAs leading to URSA.MethodsWe profiled lncRNAs expression in peripheral blood from 5 non-pregnant URSA patients and 5 matched healthy control women by lncRNA microarray analysis. Functions of URSA-associated lncRNAs were further investigated in vitro.ResultsRP11-115N4.1 was identified as the most differentially expressed lncRNA which was highly upregulated in peripheral blood of non-pregnant URSA patients (P = 3.63E-07, Fold change = 2.96), and this dysregulation was further validated in approximately 26.67% additional patients (4/15). RP11-115N4.1 expression was detected in both lymphocytes and monocytes of human peripheral blood, and in vitro overexpression of RP11-115N4.1 decreased cell proliferation in K562 cells significantly. Furthermore, heat-shock HSP70 genes (HSPA1A and HSPA1B) were found to be significantly upregulated upon RP11-115N4.1 overexpression by transcriptome analysis (HSPA1A (P = 4.39E-08, Fold change = 4.17), HSPA1B (P = 2.26E-06, Fold change = 2.99)). RNA pull down and RNA immunoprecipitation assay (RIP) analysis demonstrated that RP11-115N4.1 bound to HNRNPH3 protein directly, which in turn activate heat-shock proteins (HSP70) analyzed by protein-protein interaction and HNRNPH3 knockdown assays. Most importantly, the high expression of HSP70 was also verified in the serum of URSA patients and the supernatant of K562 cells with RP11-115N4.1 activation, and HSP70 in supernatant can exacerbate inflammatory responses in monocytes by inducing IL-6, IL-1β, and TNF-α and inhibit the migration of trophoblast cells, which might associate with URSA.ConclusionOur results demonstrated that the activation of RP11-115N4.1 can significantly increase the protein level of HSP70 via binding to HNRNPH3, which may modulate the immune responses and related to URSA. Moreover, RP11-115N4.1 may be a novel etiological biomarker and a new therapeutic target for URSA.