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. QuantitativeRT-PCRrevealed decreasedmRNAexpression 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. Asignificantly lowerlevel of H3K27me3 in molar placentawas 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, wedemonstrate the potential of c-myc as a fetal DNA epigenetic marker for pre-eclamptic pregnancies. Thuswe 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.