Beenish Rahat

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

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

The invasion cascade exhibited by placental trophoblasts and cancerous cells bears many similarities, and it is attributed to extracellular matrix degradation mediated by matrix metalloproteinases (MMPs). Although proper and controlled invasion by trophoblasts into the maternal uterus is an essential requirement for maintenance of normal pregnancy, any abnormality in this phenomenon results in the development of invasion-related disorders such as gestational trophoblastic diseases (GTDs) and preeclampsia. We studied the epigenetic basis of differential expression of two placental MMPs (MMP2 and MMP9) and tissue inhibitors of metalloproteinases (TIMP2 and TIMP1) during normal gestation and invasion-related disorders, i.e., preeclampsia and GTDs. Our study suggests the association of H3K9/27me3 with differential expression of these MMPs and their inhibitors, which regulate the placental invasion during normal pregnancy, whereas no role of CpG methylation was observed in the differential expression of MMPs/TIMPs. Further, development of GTDs was associated with abnormally higher expression of these MMPs and lower levels of their inhibitors, whereas the reverse trends were observed for MMPs and their TIMPs in case of preeclampsia, in association with abnormal changes in H3K9/27me3. These results suggest the involvement of higher levels of MMPs in an aggressive invasive behavior depicted by GTDs, whereas lower levels of these MMPs in shallow and poor invasive phenotype associated with preeclampsia. Thus, our study shows the significance of a proper balance regulated by histone trimethylation between differential expression of MMPs and their TIMPs for maintaining normal pregnancy and its deregulation as a contributing factor for pathogenesis of invasive disorders during pregnancy.

Identification of regulatory mechanisms of intestinal folate transport in condition of folate deficiency

Folic acid is an essential micronutrient, deficiency of which can lead to disturbance in various metabolic processes of cell. Folate transport across intestine occurs via the involvement of specialized folate transporters viz. proton coupled folate transporter (PCFT) and reduced folate carrier (RFC), which express at the membrane surfaces. The current study was designed to identify the regulatory mechanisms underlying the effects of folate deficiency (FD) on folate transport in human intestinal cell line as well as in rats and to check the reversibility of such effects. Caco-2 cells were grown for five generations in control and FD medium. Following treatment, one subgroup of cells was shifted on folate sufficient medium and grown for three more generations. Similarly, rats were fed an FD diet for 3 and 5 months, and after 3 months of FD treatment, one group of rats were shifted on normal folate-containing diet. Increase in folate transport and expression of folate transporters were observed on FD treatment. However, when cells and rats were shifted to control conditions after treatment, transport and expression of these genes restored to the control level. FD was found to have no impact on promoter methylation of PCFT and RFC; however, messenger RNA stability of transporters was found to be decreased, suggesting some adaptive response. Overall, increased expression of transporters under FD conditions can be attributed to enhanced rate of transcription of folate transporters and also to the increased binding of specificity protein 1 transcription factor to the RFC promoter only.

Gene specific epigenetic regulation of hepatic folate transport system is responsible for perturbed cellular folate status during aging and exogenous modulation.

SCOPE The present study was designed to identify the molecular mechanism of folate modulation and aging on aberrant liver folate transporter system. METHODS AND RESULTS An in vivo rat model was used, in which weanling, young and adult rats were given folate deficient diet for 3 and 5 months and after 3 months of folate deficiency, one group received physiological folate repletion (2 mg/kg diet) and another group received over supplemented folate diet (8 mg/kg diet) for another 2 months. In adult group, 3 and 5 months of folate deficiency decreased serum and tissue folate levels with decreased uptake of folate, further associated with decreased expression levels of reduced folate carrier (RFC) and increased expression levels of folate exporter (ABCG2) at both mRNA and protein levels, which in turn regulated by promoter hypermethylation of RFC and promoter hypomethylation of ABCG2 gene. CONCLUSION Promoter hypermethylation of RFC and promoter hypomethylation of ABCG2 may be attributed to the down regulation of RFC and up regulation of ABCG2 at mRNA and protein levels in conditions of 3 and 5 months of folate deficiency in the adult group.

Association of aberrant methylation at promoter regions of tumor suppressor genes with placental pathologies.

AIM The resemblance between invasive behavior of cancer cells and placental trophoblasts and the role of aberrant epigenetic regulation in cancer development is well known. METHODS We analyzed the role of promoter region CpG-methylation and H3K9/27me3 of tumor suppressor genes in normal and pathological pregnancies and utilized their CpG-methylation data to search for fetal DNA epigenetic marker in maternal blood. RESULTS CpG and H3K9/27-methylation associated decreased expression of RASSF1A and APC and increased expression of P16, RB1 and PRKCDBP was observed with advancing normal gestation. Gestational trophoblastic diseases and preeclampsia revealed gene-specific epigenetic deregulation of candidate tumor suppressor genes. Furthermore, APC and PRKCDBP showed the potential to act as fetal DNA epigenetic markers, similar to RASSF1A. CONCLUSION Deregulation of methylation of tumor suppressor genes contributes to the development of preeclampsia and gestational trophoblastic diseases. APC and PRKCDBP may act as fetal DNA epigenetic markers for prenatal diagnosis.

Epigenetic modifications at DMRs of placental genes are subjected to variations in normal gestation, pathological conditions and folate supplementation.

Invasive placentation and cancer development shares many similar molecular and epigenetic pathways. Paternally expressed, growth promoting genes (SNRPN, PEG10 and MEST) which are known to play crucial role in tumorogenesis, are not well studied during placentation. This study reports for the first time of the impact of gestational-age, pathological conditions and folic acid supplementation on dynamic nature of DNA and histone methylation present at their differentially methylated regions (DMRs). Here, we reported the association between low DNA methylation/H3K27me3 and higher expression of SNRPN, PEG10 and MEST in highly proliferating normal early gestational placenta. Molar and preeclamptic placental villi, exhibited aberrant changes in methylation levels at DMRs of these genes, leading to higher and lower expression of these genes, respectively, in reference to their respective control groups. Moreover, folate supplementation could induce gene specific changes in mRNA expression in placental cell lines. Further, MEST and SNRPN DMRs were observed to show the potential to act as novel fetal DNA markers in maternal plasma. Thus, variation in methylation levels at these DMRs regulate normal placentation and placental disorders. Additionally, the methylation at these DMRs might also be susceptible to folic acid supplementation and has the potential to be utilized in clinical diagnosis.

Reduced SP1-mediated transcriptional activation decreases expression of intestinal folate transporters in response to ethanol exposure

SCOPE The study was designed to identify the regulatory mechanisms underlying the effects of ethanol exposure on intestinal folate transport and to investigate the reversibility of such effects. METHODS AND RESULTS Caco-2 cells were grown in control and ethanol containing medium for 96 h. Thereafter, one subgroup of cells was shifted on ethanol free medium and grown for next 72 h. For in vivo studies, rats were given 1g ethanol/kg body weight/day either for 3 or 5 months and after 3 months of ethanol treatment, one group of rats received no ethanol for 2 months. A significant decrease in folic acid transport as well as expression of folate transporters was observed on ethanol treatment and the effects were reversible upon removal of ethanol. Ethanol exposure had no impact on CpG island methylation of the folate transporters however, an increase in their mRNA half-life was observed that seems to be a homeostatic mechanism. Chromatin immunoprecipitation assay revealed a decrease in binding of SP1 transcription factor to the promoter regions of folate transporters. CONCLUSION Reduced binding of SP1 to the promoter region of folate transporters may be a part of the regulatory mechanism resulting in decreased expression of folate transporters on ethanol exposure.

Epigenetic regulation of STAT5A and its role as fetal DNA epigenetic marker during placental development and dysfunction.

INTRODUCTION Development of normal placenta requires regulated apoptosis of trophoblasts. However, uncontrolled apoptosis has been seen in the pregnancy related complications like hydatidiform mole and pre-eclampsia. STAT5A is a transcription factor with well-known anti-apoptotic role. Thus, we sought to study the role of STAT5A and its epigenetic regulation in placental development and pathologies and its use as fetal DNA epigenetic marker. METHODS The present study was conducted on pregnant women who were enrolled in five groups, based on the three trimesters in normal pregnancy and two pregnancy related disorder groups: pre-eclampsia and hydatidiform mole. Placental villi samples and maternal blood were obtained from each pregnant woman and were analyzed for promoter region methylation (via methylation sensitive high resolution melting) and histone trimethylations (via chromatin immunoprecipitation) of STAT5A. RESULTS Our data revealed higher expression of STAT5A in first trimester villi, which decreased with advancing gestation with corresponding increased DNA methylation and H3 trimethylations. Development of choriocarcinoma was associated with DNA methylation associated lower expression of STAT5A. The pattern of promoter methylation of STAT5A in cell free DNA within maternal plasma was observed to be similar to its promoter methylation in placental villi during normal pregnancy, pre-eclampsia and molar complications, which suggested its use as a novel fetal DNA epigenetic marker. DISCUSSION Our results suggest the regulation of STAT5A via epigenetic mechanisms during normal pregnancy and the association of STAT5A epigenetic dysregulation in pregnancy related complications. Further, hypermethylated STAT5A can be utilized as novel fetal DNA epigenetic marker.

Microsomal Epoxide Hydrolase Polymorphisms and Haplotypes as Determinants of Hepatitis B Virusand Hepatitis C Virus-related Liver Disease in Indian Population

BACKGROUND Hepatocellular carcinoma (HCC) is the fifth most common cancer and third leading cause of death worldwide. Main causes of HCC are hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. mEPHX, a xenobiotic metabolizing enzyme, exhibits a dual role of procarcinogen detoxification and activation, hence considered as a cancer risk factor as well as a protective factor. Two known polymorphic forms of mEPHX, exon in exon 3 and 4, are associated with the development of HCC. OBJECTIVE To determine the association of genotypes and haplotypes of mEPHX with risk of HCC developments separately in HBV- and HCV-infected carriers and patients with hepatitis. METHODS Polymerase chain reactions (PCR) were carried out using primers to amplify exon 3 (113 Tyr→His variant) and exon 4 (139 His→Arg) polymorphic sites. To distinguish the wild and variant forms, PCR amplification products were digested with restriction endonucleases EcoRV and Rsa1 for exons 3 and 4, respectively. RESULT Exon 3 genotypes, Y113H and H113H, shared a protective association with HBV-chronic hepatitis infection (P < 0.001 and P< 0.01, respectively) as well as HBV-HCC development (P < 0.001) among HBV-carrier group, while Y113H acts as a risk factor for HCV-chronic hepatitis development (P < 0.001) as well as for HCC development (P < 0.01) with HCV-carrier group as reference. Both H139R and R139R, exon 4 genotypes, acted as a risk factor for HBV/HCV-chronic hepatitis infection and for HBV/HCV-HCC development (P ranges from < 0.05 to < 0.001) with HBV/HCV carriers as reference. 113His-139His and 113His-139Arg haplotypes shared a significant negative and positive association, respectively, with HBV hepatitis and HBV-HCC risk. 113Tyr-139Arg haplotype acted as a risk for HCV-HCC development. CONCLUSION Polymorphic and haplotypic variant forms of mEPHX exon 3 and 4 variably determine the susceptibility to develop HCC in HBV- and HCV-carrier subjects.

Modulation of dietary folate with age confers selective hepatocellular epigenetic imprints through DNA methylation

The present study has been designed to determine the effect of folate modulation (deficiency/supplementation) with aging on the promoter methylation of tumor suppressor and proto-oncogenes to understand the underlying mechanism of epigenetic alterations. Folate deficiency was induced for 3 and 5 months in weanling, young and adult groups, and after 3 months of folate deficiency, they were repleted with physiological folate (2 mg/kg diet) and folate oversupplementation (8 mg/kg diet) for another 2 months. The methylation facet in the present study revealed that the combined effect of folate deficiency and aging decreased the methylation index. Folate deficiency with age resulted in the up-regulation of proto-oncogenes (C-MYC and C-JUN) and cell cycle regulator gene Cyclin E as a result of promoter hypomethylation. However, in case of tumor suppressor genes (p53, p15ink4b and p16ink4a), the expression levels were found to be decreased at transcriptional level due to promoter hypermethylation. Upon repletion with physiological folate and folate oversupplementation, we found down-regulation of proto-oncogenes and up-regulation of tumor suppressor genes as a result of promoter hypermethylation and hypomethylation, respectively. Deregulation of these important genes due to folate deficiency may contribute toward the pathogenesis at cellular level.