Past Issue

Volume 9, Supplement 1, Summer 2015 (Presented at 16th Congress on Reproductive Biomedicine and 10th Royan Nursing and Midwifery Seminar) Pages: 42-42

O-37: Pseudomalignant Nature of Placenta during Normal and Pathological Gestation Is Regulated by Epigenetic Mechanisms which Can be Exploited To Design Non-Invasive Fetal Dna Markers

Placentation shares many analogues with the development of tumors such as rapid proliferation, invasiveness, gene expression profiles especially the expression of tumor suppressor genes, oncogenes and matrixmetallo proteinases (MMPs). Thus, a placenta has been described as a pseudomalignant tissue. However, placentation is tightly regulated and any deregulation of this pseudomalignant nature leads to the development of gestational trophoblastic diseases (GTDs) and preeclampsia. Therefore, this study was designed to analyze the role of epigenetic mechanisms like DNA methylation and histone3 trimethylation at promoter regions of tumor suppressor genes (RASSF1A, APC, P16, RB1 and PRKCDBP), oncogenes (c-myc, c-jun, VEGF, EGFR and hTERT) and MMPs (MMP-2 &-9) and tissue inhibitors of MMPs (TIMP-2 &-1) in maintaining normal pregnancy and pathogenesis of pregnancy related disorders. Further, the methylation data was utilized to search for novel fetal DNA epigenetic markers in maternal plasma.
Materials and methods
Epigenetic profiling was done in five subject groups, which included first, second and third trimester with normal pregnancy and preeclampsia and hydatidiform mole complicated pregnancies (n=30 for each group) and a choriocarcinoma cell line (JEG-3). Placental villi sample and maternal blood were collected from each pregnant woman. DNA methylation and Histone3K9/27me3 were estimated via Methylation-Specific High Resolution Melting (MS-HRM) and chromatin immunoprecipitation assay (ChIP) respectively, while mRNA expression was quantified by qRTPCR.
Our study revealed the association of advancing normal gestation with decreasing expression of RASSF1A and APC mediated by DNA methylation and H3K9/27me3, while increasing expression of P16, RB1 and PRKCDBP regulated by H3K9/27me3 only for P16 and RB1, while by H3K9/27me3 and DNA methylation for PRKCDBP within normal placental villi samples. CpG methylation independent and H3K9/27me3 mediated reverse trend was observed in the differential expression of MMP-2 & -9 and their inhibitors (TIMP-2 & -1 respectively) in regulating the placental invasion during normal pregnancy. Decreasing expression levels of VEGF, EGFR, c-myc, c-jun and hTERT with advancing normal gestation, was independent of DNA methylation, while mediated by H3 K9/K27 trimethylations. Development of preeclampsia was observed to be associated with decreased expression of all studied oncogenes, MMPs, RASSF1A and P16 and increased expression of TIMPs and PRKCDBP. However, development of GTDs were observed to be associated with increased expression of oncogenes, MMPs and decreased expression of tumor suppressor genes and TIMPs. These changes were associated with abnormal changes in DNA methylation and H3K9/27me3. Further, based on difference in methylation of these genes of placental and maternal origin, we have been able to report few fetal DNA epigenetic markers in maternal plasma, to be utilized for prenatal diagnosis like APC, PRKCDBP and for specific diagnosis of pregnancy related disorders like c-myc and VEGF.
Thus, our study is the first of its kind to report the role of DNA and H3 methylation in regulating the pseudomalignancy during normal gestation and dysregulation of these mechanisms as contributing factors for development of placental disorders. Further, we have reported a few novel fetal DNA markers for non-invasive prenatal diagnosis and specific diagnosis of pregnancy related disorders.