(188) The N6-Methyladenosine (m6A) Post-Transcriptional Modification Alters RNA and Protein Expression of EMT Markers in Trophoblast Cells 

Petra. A. Rose1 and Jennifer. R. Wood1 

1. Department of Animal Science, University of Nebraska-Lincoln, Lincoln, NE, USA


Abstract Text: Maternal obesity contributes to adverse maternal and congenital outcomes, due in part to placental abnormalities. Our previous RNA sequencing of mid-gestation mouse placentas from obese dams, predicted global downregulation of differentially expressed genes (DEGs) compared to metabolically normal dams. Further, qPCR analysis confirmed low abundances of genes that encode epithelial mesenchymal transition (EMT) markers in the placental junctional zone. Since migratory trophoblast lineages are housed in the junctional zone, this suggested that obesity alters the EMT process during placentation; however, the mechanism remains elusive. While most studies focus on the transcriptional regulation of DEGs, much less is known about post-transcriptional regulation of mRNA stability and translation. RNA methylation at the N6-methyladenosine (m6A) is a common, reversible post-transcriptional modification. Methyl groups are added to an RNA by a methyltransferase complex that includes METTL3. Conversely, methyl groups are removed from an RNA by demethylases including FTO. As the methylation status changes, RNA binding proteins (i.e., readers) regulate storage, degradation, and translation of the transcript. The objective of this study was to investigate how enhancing or inhibiting RNA methylation using either METTL 3 or FTO inhibitors would alter the abundance of genes related to trophoblast differentiation and migration. To accomplish this objective, we treated JAR cells, which is a human choriocarcinoma cell line, with either STM2457 or FB23-2. STM2457 inhibits METTL3 and therefore reduces RNA methylation while FB23-2 inhibits FTO resulting in increased RNA methylation. The cells were maintained in media containing high-glucose DMEM and 10% FBS. To perform the experiment, JAR cells were serum starved prior to treatment with 10-20 mM FB23-2 (n=8), or 10-20 mM STM2457 (n=8) for 48 hours. Serum-starved untreated cells (n=4) served as control. Post-treatment, cells were collected and either RNA or protein isolated. Quantitative, real-time PCR was performed using reverse-transcribed cDNA from control and treated cells and primers designed against EMT markers and previously identified DEGs. When cells were treated with STM2457, the abundance of SNAIL1 (8-fold), SNAIL2 (62-fold), CEBPA (42-fold), and FOXA2 (24-fold) transcripts were significantly increased compared to control. In addition, treatment of cells with FB23-2 significantly decreased FOXA2 (12-fold) mRNA abundance. To determine how treatment with STM2457 and FB23-2 affected protein expression, Western blot analysis was performed using an antibody against CEBPA. While there was no effect of FB23-2 on CEBPA levels, STM2457 significantly reduced CEBPA levels (2-fold) compared to control. These data suggest that inhibition of RNA methylation (i.e., reduced m6A) blocked translation of the CEBPA transcript resulting in its increased abundance. Furthermore, we propose that RNA methylation plays an important role in trophoblast cell differentiation and/or migration mediated by EMT transcription factors.