Epigenetic mechanisms regulate placental c-myc and hTERT in normal and pathological pregnancies; c-myc as a novel fetal DNA epigenetic marker for pre-eclampsia

Abstract

Placental development is known for its resemblance with tumor development, such as in the expression of oncogenes (c-myc) and telomerase (hTERT). The expression of c-myc and hTERT is up-regulated during early pregnancy and gestational trophoblastic diseases (GTDs). To determine the role of DNA methylation [via methylation-sensitive high resolution melting (MS-HRM)] and histone modifications [via chromatin immunoprecipitation (ChIP assay)] in regulating the differential expression of c-myc and hTERT during normal gestation and their dysregulation during placental disorders, we obtained placental samples from 135 pregnant women, in five groups: normal first, second and third trimester (n = 30 each), pre-eclamptic pregnancy (n = 30) and molar pregnancy (n = 15). Two placental cell lines (JEG-3 and HTR-8/SVneo) and isolated first-trimester cytotrophoblasts were also studied. Quantitative RT-PCR revealed decreased mRNA expression levels of c-myc and hTERT, which were associated with a higher level of H3K9me3 (1.5-fold, P < 0.05) and H3K27me3 (1.9-fold, P < 0.05), respectively, in third-trimester placental villi versus first-trimester villi. A significantly lower level of H3K27me3 in molar placenta was associated with a higher mRNA expression of c-myc and hTERT. The development of pre-eclampsia (PE) was associated with increased methylation (P < 0.001) and H3K27me3 (P < 0.01) at the c-myc promoter and reduced H3K9me3 (P < 0.01) and H3K27me3 (P < 0.05) at the hTERT promoter. Further, mRNA expression of c-myc and hTERT was strongly correlated in molar villi (r = 0.88, P < 0.01) and JEG-3 cells (r = 0.99, P < 0.02). Moreover, on the basis of methylation data, we demonstrate the potential of c-myc as a fetal DNA epigenetic marker for pre-eclamptic pregnancies. Thus we suggest a role for epigenetic mechanisms in regulating differential expression of c-myc and hTERT during placental development and use of the c-myc promoter region as a potential fetal DNA marker in the case of PE.

Imbalance between matrix metalloproteinases and their tissue inhibitors in preeclampsia and gestational trophoblastic diseases

“…Earlier studies have reported the expressions of MMP2 and MMP9 within the first-trimester placenta (StaunRam & Shalev 2005), whereas high expression levels of MMP2 and MMP9 have been observed in highly invasive extravillous trophoblasts (Seval et al 2004). Our previous study (Rahat et al 2014) and few recent studies (Dokras et al 2006, Rahnama et al 2006 have highlighted the importance of epigenetic mechanisms in the regulation of gene expression during placentation. Therefore, we aimed to find the significance of differential mRNA expressions and the epigenetic regulatory mechanisms of MMP2 and MMP9 and their inhibitors in the development of pathological pregnancies, with reference to normal physiological pregnancies.…”

“…Cytotrophoblasts were isolated from first-trimester placentas (6-11 weeks) by enzymatic digestion of first-trimester villi as explained previously (Rahat et al 2014). Briefly, 2 g dissected first-trimester villi was subjected to sequential trypsin digestion.…”

“…Total genomic DNA isolated by DNA isolation kit (Real Genomics, Real Biotech Corporation, Taipei, Taiwan), from placental villi samples, maternal blood leukocytes, JEG-3 cell line, and circulating DNA isolated by Miniprep DNA isolation kit (Bioserve, Beltsville, MD, USA), from maternal plasma, was used to estimate the promoter region methylation percentage by methylation-sensitive high-resolution melting (MS-HRM) as described previously (Rahat et al 2014). Briefly, DNA methylation standards were prepared for their use in the estimation of DNA methylation of study samples.…”

“…The chromatin immunoprecipitation (ChIP) assay was performed only on placental villi samples to estimate the levels of H3K9me3 and H3K27me3 at promoter region of genes as described previously (Rahat et al 2014). Briefly, 25 mg placental villi were fixed in 1.5% formaldehyde to cross-link histones with DNA, followed by its disaggregation into single-cell suspension.…”

Increased synthesis of folate transporters regulates folate transport in conditions of ethanol exposure and folate deficiency

“…Thereafter, MS-HRM was performed as described by Rahat et al [30]. In order to prepare standard curve for the determination of percentage methylation of the CpG islands present within the promoter region of folate transporters, methylated standards were used that ranges from 0 to 100 %.…”