Pregnancies with PE [16?9], while multivitamin supplements containing folic acid and vitamin B12 are correlated with reduced risk of PE [17,19], which indicate the potential role of DNA methylation in the pathophysiology of PE. Furthermore, to acquire 
22948146 a deep insight into PE at the molecular level, the association of DNA methylation with PE has been intensively studied both in the gene-specific pattern [20?2] and in the genome-wide level [17,23]. In addition to the well-defined SERPINA3 [22], APC [21] and TIMP3 [23,24], a series of genes with differential methylation related to collagen metabolism, angiogenesis and blood vessel development have also been identified to be involved in PE with aberrant methylation patterns [17,25]. Given all that, DNA methylation appears to be a significant factor in the development of PE.Upregulation and Hypomethylation of Genes in PEThe causal relationship between promoter DNA methylation aberrations and gene expression differences has been well established. Promoter hypermethylation has been shown to associate with transcriptional repression and hence decreases expression and functional gene dosage; 22948146 a deep insight into PE at the molecular level, the association of DNA methylation with PE has been intensively studied both in the gene-specific pattern [20?2] and in the genome-wide level [17,23]. In addition to the well-defined SERPINA3 [22], APC [21] and TIMP3 [23,24], a series of genes with differential methylation related to collagen metabolism, angiogenesis and blood vessel development have also been identified to be involved in PE with aberrant methylation patterns [17,25]. Given all that, DNA methylation appears to be a significant factor in the development of PE.Upregulation and Hypomethylation of Genes in PEThe causal relationship between promoter DNA methylation aberrations and gene expression differences has been well established. Promoter hypermethylation has been shown to associate with transcriptional repression and hence decreases expression and functional gene dosage; 1655472 while demethylation in the promoter region will increase the transcription. Given the association of DNA methylation with PE mentioned above, we want to make an investigation of genes and functional networks with regard to epigenetics. So far, comparative gene expression profiling analyses of normal and pathological placentas have identified subsets of genes with altered expression [26]; however, few studies have been performed to explore the mechanisms that underlie the differential gene expression. Consequently, it is valuable to estimate the relative contribution of changes in DNA methylation levels to gene expression differences. In the current study, we have performed a global gene expression profiling via oligonucleotide microarrays of placental tissue from preeclamptic pregnancies and uncomplicated pregnancies to explore genes with differential expression. Among the differentially expressed genes in our study, we picked up LEP, the gene with biggest expression difference, and SH3PXD2A, the gene with the most significant p value, for further analysis to explore the relevance of DNA methylation in the development of PE as increasing studies suggested that aberrant DNA methylation was considered as a pathogenic factor in the onset of PE [20,27,28].Table 1. Clinical characteristics of the study population.PE (n = 23)a 30.9566.48 35.3463.07 30.7263.49 154.18617.85 106.27615.07 4.0664.63 2281.56969.90 402.66143.Characteristic Maternal age (years) Gestational age (weeks) Pregnancy BMI (kg/m ) Systolic BP (mmHg) Diastolic BP (mmHg) Proteinuria (g/24 h) Infant birthweight (g) Plcenta weight (g)Control (n = 22) 28.563.73 39.3861.19 31.4563.76 108.56612.84 68.6968.93 0 34066293.83 692.14674.p-valueb0.387 1.0261025 0.616 8.99610211 8.99610211 1.55610210 2.9461024 2.All results are presented as mean 6 SD. a Diagnostics criteria used for PE patients were as follows: systolic pressure .140 mmHg, diastolic pressure .90 mmHg, and proteinuria .0.3 g in a 24 hour collection. b Obtained using the Mann-Whitney U-test on SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). doi:10.1371/journal.pone.0059753.tRNA PreparationTotal RNAs were extracted from placentas using the mirVanaTM miRNA Isolation Kit (Ambion) according to manufacturer’s instruction, including a DNase I digestion step. The quality of RNA was determined by Nanod.
