Bioinformatics Analysis Of Differentially Expressed Genes In Preeclamptic Placental Tissues

1、Application ofcDNAmicroarrayscreeningdifferentially expressed genesin placenta of preeclampsia cases and Normal controls Background:,preeclampsia-eclampsiais apregnancyidiopathic disease,etiologyand pathogenesishas not yet beenfully elucidated.It is one of theimportant research topic of Obstetric.The results of a large number ofstudiessuggest thatpreeclampsia may becaused byboth geneticand environmental factors.Abnormalplacentaplays an important roleinthe pathogenesis ofpre-eclampsia.With thein-depthstudyofthe humangenome,susceptibility geneshas become a focus in geneticsresearch ofpreeclampsia.Materials and methods:Selectpreeclampsia and normal controled pregnant women each10cases,in August,2012,inShandong Provincial Hospitalobstetric.Cesarean deliveriesage, parity, gestational agematched.Extractionand processing RNA fromplacental tissue;preparedthefirst-strand ofcDNAprobes;Purificationandquantitativefluorescent probe.Preparation ofmicroarrays,Use the gene chip （cDNA microarray）, BIOSTARH140s （the Shanghai united gene company）, Chip hybridization, washing.Use ScanArray4000scanner （Packard Biochip Technologies, Inc., USA） scaned chips.Obtained the data of experiment. For data normalization processing and quality control.Gene chip hybridization results:Obtained the results of cDNA expression profile chip hybridization, add positive and negative two-way tag samples of preeclampsia patients and normal controls.Fluorescence exchange treatment to avoid the false positive rate caused by nonspecific fluorescence adsorption, The results show that the experimental results for the first time and second experimental results after swap fluorescent tags, respectively for382and394differentially expressed genes111common differentially expressed genes in the both two experiments, percentage of29.1%and28.2%respectively. In111differentially expressed genes, in preeclampsia group,raised genes is68（the ratio over2.0）,downgrade express gene has44（ratio of0.5or less）. In MatchMiner software processing, obtaining GENEBANK ID.2、Bioinformatics analysis of differentially expressed genes in preeclampsia placenta tissue obtained by cDNA microarrayMethods:using DAVID （The Database for The Annotation, The Visualization andlntegrated Discovery） analysis tools to analysis differentially expressed genes of preeclampsia placenta tissue, in gene ontology （GO） analysis and the analysis of enrichment.Results:Functional clustering tool to obtain two sets of genes enriched which fraction is greater than two. Functional annotation function annotation tools were the following analysis. Functional Annotation Chartanalysis results:A total of86significant functional GO classification items, including715differentially expressed genes. In the molecule,11GO terms in gene function （FDR<0.05）,had activity relationship with active structure of the molecule and the catalytic activity. In a biological process, related with biological adhesion, cellular processes, metabolic processes, structure, area construction and structural areas. In cell components,related with the extracellular region and part of the extracellular region. Functional annotation clustering also drew conclusions point to glycoprotein, signal, signal peptide, disulfide bonds, and part of the extracellular region of the extracellular region,as functional annotation map. Functional annotation table input genes to make a functional annotation one by one. Finally,16kinds of target gene relatedto pre-eclampsia was selected.3、Using real-time quantitative PCR method to validated preeclampsia placenta differentially expressed genes.Theoretical basis:Real-time PCR principle is added fluorophores in the PCR reaction system, using real-time monitoring to detect fluorescence signals accumulated PCR process, amplification process is monitored in real time, fluorescence signal associated with amplificationanalyses continuously. Monitor changes in the fluorescence signal with the reaction time can be plotted as a curve. Finally, we have a standard curve by a mathematical formula for the unknown template for quantitative analysis, use standard curve or mathematical formula to calculate the number of copies of the initial sample.Materials and Methods:16selected candidate genes by Real-time PCR method to observe the changes in mRNA levels. Clinical data:same as before. Total RNA was extracted from placental tissue,synthesis of primers, RNA was reverse transcribed according to the reaction system supplied with the kit for reverse transcription reaction. Quantitative PCR reactions to generate a standard curve.Results:Real Time-PCR to detect differential expression of placenta tissue and normal placental tissue, which has three falling tone,13up-regulated. Comparison with the results of gene chip, both methods have a good correlation, and16genes both consistent with the direction of change, and closed to the level of gene expression.4、Results and discussion:Bioinformatics analysis revealed that the differentially expressed genes play an important role in preeclampsia.Including:（1）、latent transforming growth factor Pbinding protein2（NM₀00428）、Insulin-like growth factor binding protein1（CR595377）;（2）Immune-related factors:Interferon receptor1（NM₀00629） Mannose receptor C1（NM₀02438）;（3）Pregnancy-associated protein （AF342989、 M23575、BC063127）;（4）Cell signaling pathway related genes:（AK129833、 BC012609）;（5）Other:Cytokeratin （AK122864）、Fragile X syndrome gene （BC067272） CytochromeP450（NM₀31226）。The results were analyzed and discussed, drew the following conclusions:1, There are significant differences between genes expressed of preeclampsia and normal placental tissue, indicating the presence of metabolic imbalance in preeclampsia placenta.2, Gene chip technology is efficient and fast tool to screening differentially expressed genes in placenta of preeclampsia, real-time quantitative RT-PCR can be expressed as a single genetic change research, both them complementary and verification.3, Enrichment analysis of gene function draw a significant association, with low false positive rate and the function categories of targeting of gene.4, some abnormal gene expressed in preeclampsia placenta tissue indicating that it may be involved in the pathogenesis of preeclampsia, it may be the result of pathophysiological changes of preeclampsia.5, the potential of transforming growth factor-P binding protein2（LTBP-2） expression abnormalities reduce, inhibit trophoblast invasion, spiral artery recast incomplete, causing placental ischemia and hypoxia, followed by activation and vascular endothelial cells, promote preeclampsia-eclampsia.6, insulin-like growth factor binding protein1（IGFBP1） abnormal expression increased, leading to erosion barriers trophoblast cells, placenta shallow implantation, causing preeclampsia7, pregnancy-specific proteins including pregnancy-associated plasma protein A （PAPP-A）, pregnancy-associated plasma protein B （PAPP-B） and pregnancy-associated plasma protein C （PAPP-C） that pregnancy-specific β1-glycoprotein （PSG）, abnormally elevated expression, inhibition of maternal rejection, prolonged gestation time to ensure fetal survival.8, mannose receptor family gene （mannose receptor gene1, MRC1） regulating the replication fork in S phase separation polymerization, DNA replication, interference genetic stability genes.In this study, the difference in preeclampsia placenta tissue by microarray gene expression studies, found111differentially expressed genes, bioinformatic analysis by DAVID software, selected16seeds preeclampsia placenta plays an important role in gene and real-time fluorescence quantitative RT-PCR validation for finding placenta-related genes provide clues. However, the incidence of preeclampsia and complex factors, especially diverse clinical manifestations in patients with severe preeclampsia, there are individual differences among patients, to determine the significance of differentially expressed genes and study the interaction may exist between the various genes is still pending in a more comprehensive, in-depth research.