An Apparent Mechanism Of Environmental Factors On The Effects Of Environmental Factors On Fetus Of Cardiac

Heart development is a highly complex, spatio-temporal morphological process, which is easy to be disturbed by environmental factors that can lead to cardiac defects. It is believed that environment as a factor is contributed to the occurrence of 80% of cardiac defects. Environmental factors include maternal exposure to external environmental conditions, like toxic substances attack and microenvironment abnormalities in the maternal body, like maternal diseases, nutritional lack of some multivitamins, protein, fat during the pregnancy. Environment as a factor is involved in the formation of plastic phenotype. During the process epigenetic mechanism is helpful. Epigenetic mechanism is a mark when environment affect the development of various organs in animals and humans. And epigenetic modifications are heritable, contributing to the pathogenesis of some kinds of disease in offspring. So we postulate that the impact of adverse environmental exposure on organ development and phenotypic alterations during the pregnancy is associated with epigenetic modifications. We plan to investigate the role of epigenetic modifications in the pathology of cardiac defects through DNA methylation. We study epigenetic mechanism in cardiac tissue in both normal and abnormal condition.In our study, there are three parts, covering DNA methylation, genomic imprinting and microRNA, which are 1) The regulation mechanism of DNA methylation status in cardiac defects; 2) The impact of pregnant alcohol intake on methylation status of imprinting genes in fetal mouse brain, heart and placenta tissue; 3) Priliminary study of microRNA expression profiling during fetal cardiac developmentPart1 The regulation mechanism of DNA methylation in cardiac defectsObjective:to analyze the global methylation level and gene-specific methylation level as well as alterations of the gene expression in cardiac tissue from cardiac defects.Materials and methods:We collect gestational-week matched cardiac tissue from a total of 17 cases of isolated cardiac defects,14 cases of un-isolated cardiac defects and 22 cases of normal fetal hearts. After DNA extraction, bisulphite treatment, we applied Roche NimbleGen’s DNA methylation array in 5 cases of cardiac defects (2 cases of iso-cardiac defects and 3 cases of non-iso cardiac defects) and 4 cases of normal controls, acquired CpG sites profile with differential methylation level. Then in a larger sample size of 15 cases of iso-cardiac defects,11 cases of non-iso cardiac defects and 18 cases of normal controls, applying Sequenom MassARRAY EpiTYPER platform, we quantitatively test the methylation level of 10 gene-specific CpG sites which is related to heart development. Subsequently, throught q RT-PCR and Westernblot, we analyze the expression of EGFR、SMAD7、SLC19A1、 NOTCH1 genes in 7 cases of iso-cardiac defects and normal controls, respectively. Among 10 gene-specific CpG sites, intragenetic CpG sites of NOTCH1 had a lower methylation level, while intergenetic CpG sites of EGFR, intragenetic CpG sites of SMAD7, intergenetic CpG sites of SLC19A1 had a higher methylation level in both isolated and non-isolated cardiac defects. EGFR gene had a lower expression trend at mRNA and protein level; SMAD7 gene had a high expression trend at mRNA and protein level; SLC19A1 gene had a statitically significantly lower mRNA expression and lower expression trend at protein expression. NOTCH1 gene had a lower mRNA expression, but a uncertain protein expression.Conclusions:Global DNA methylation status, particularly gene-specific methylation status and expression of EGFR、SMAD7、SLC19A1、NOTCH1 genes could be involved in the pathological of cardiac defects.Part 2 The impact of prenatal exposure to alcohol intake on methylation status of Igf2/H19 imprinting gene in fetal mouse heart, brain and placentaObjective:To investigate the impact of pregnant exposure to alcohol intake on methylation status of Igf2/H19 imprinting gene in fetal mouse heart, brain and placenta.Materials and methods:Pregnant mouses were treated with alcohol lavage from 6.5 days to 15.5 days, then dissected at 16.5 days and immediately collected fetal heart, brain (mostly telencephalon) and placenta tissue. Applying Sequenom MassARRAY EpiTYPER paltform we analyzed methylation status of four DMRs in Igf2/H19 imprinting gene in fetal heart, brain and placenta, respectively. Using quantitative real-time PCR we detected the mRNA expression of Igf2 gene.Results:For mouse exposed to alcohol, in fetal hearts, methylation level of DMR1 in Igf2/H19 imprinting gene altered with a decreasing expression of Igf2 gene; in fetal brain, DMR2 altered with an obvious increasing Igf2 gene expression; in placenta, H19 DMR alters with an increasing Igf2 gene expression.Conclusions:The adverse impact of pregnant alcohol intake could be regulated by Igf2 gene, which is mediated by methylation of Igf2/H19 imprinting gene DMRs.Part 3 Priliminary study of microRNA expression profiling during fetal cardiac developmentObjective:To study the role of miRNAs during human fetal heart developmentMaterials and methods:miRNAs expression profiling from human fetal heart tissue at 5,7,9, and 23 weeks of gestation were obtained employing Affymetrix microarrays from CapitalBio Corporation. Then, differentially expressed miRNAs during human heart development were compared between any two time points using linear regression by R limma package algorithms. Cluster analysis was conducted with cluster 3.0 software applying hierachical clustering algorithm. Function analysis of miRNAs in cluster 1 and 5 was carried out throught GO analysis.Results:A total of 703 miRNAs were detected in human fetal heart. Among these miRNAs,288 differentially expressed miRNAs presented 5 different clusters with 5 different changing expression trends. miRNAs within cluster 1 and 5 were shown to target genes vital for heart development and to be involved in heart structure formation, cardiac-associated cellular events, and development-associated signaling pathways.Conclusions:We here describe the first set of miRNAs expression patterns in human fetal heart tissue. We found these patterns to be remarkably specific, suggesting specific roles for miRNAs in distinct clusters.