Screening Of Differentially Expressed MiRNA And Bioinformatics Analysis Of Peripheral Blood And Placenta In Preeclampsia

Objectives:This study intends to use high-throughput sequencing to screen microRNAs in the peripheral blood and placental tissues of pregnant women with preeclampsia and use bioinformatics analysis methods to accurately compare the differential expression profiles of the miRNAs between the samples and to select miRNAs that have differentially expressed significantly.Selection of significantly different miRNAs for target gene prediction,analysis of functional categories and associations of genes with corresponding gene ontology?GO?.Focus on the analysis of the distribution of target genes and determine the biological function of differential miRNA in preeclampsia.Materials and methods:1)Collect 3 cases of peripheral blood and 3 cases of placenta from normal pregnant and preeclampsia,that are similar age and parity of deliveries in sichuan province people's hospital,3 patients a total of 12 cases,formed the control group of normal and disease.2)Separation of serum from the collected whole blood,qiagen method is used to extract total RNA,total RNA was connected to the 5?linker and the 3?linker,followed by PCR amplification,obtain fragments of about 18-40nt using gel electrophoresis for cDNA library;Using Illumina HiSeq^TM 2500 for high-throughput sequencing,preliminary filtered to get clean sequence?the clean reads?,on the sequence length distribution of common sequence of the statistics and sample statistics,classifying the clean reads comments,then get a sample of all kinds of composition and expression of sRNA quantity information.3)Compare the obtained sequencing data with the miRNA bioinformatics database?miRBase database?version 21??to obtain the expression information of the existing miRNA in the database,including the structure,length,expression level,etc.,and calculate the expression amount.4)To determine the differential expression of miRNAs between samples,edgeR was used to analyze the significance of miRNA differences between the groups and the P value was calculated.Take P<0.01 as a high significant difference,P<0.05 as a general significant difference.5)Through bioinformatics analysis,the miRNAs of differentially expressed expression were screened,and the candidate miRNAs target genes were predicted and screened by various software such as TargetScan,PicTar,miRDB and Tarbase.The different target genes were analyzed by GO function enrichment analysis and KEGG pathway enrichment.Gene enrichment analysis was performed by using GESA software?v3.0?.The results of the GSEA analysis are explained using the Ggplot2 package.David database?v.6.8?was implemented to analyze functional categories and the association between genes and corresponding gene ontology?GO?.Results:1.Screening of serum differential miRNAs in preeclampsia:There were 725 differentially expressed mirnas between the two groups,with 343miRNA expression up-regulated and 382 miRNA expression down-regulated.There were 41 between<P<0.05,and 3 of P<0.01?mir-455-3p,mir-103a-2-5p,mir-449c-5p?.2.Screening of differential miRNAs in the placental tissues of preeclampsia:There were a total of 180 differentially expressed miRNAs in the two groups of samples,and there were 27 of them with P-values between 0.01⁰.05,and 24 P-values less than 0.01.Of these,90 are up-regulated and 90 are down-regulated.There are 25significant differences.3.Target gene prediction and bioinformatics analysis:The above three significantly upregulated miRNAs?miR-455-3p,miR-103a-2-5p,miR-449c-5p?and three significantly downregulated miRNAs?miR-518e-5p,miR-523-5p,miR-519b-5p?were selected.Using TargetScan,PicTar,miRDB,and Tarbase software to predict and screen candidate miRNAs target genes,resulting in 11,409,158,7,7,20 candidate target genes,and the common target genes are 4,2,3,3,3,3.Among them,miR-455-3p,miR-103a-2-5p,miR-4532,miR-151a-5p,miR-151b,and miR-451a were randomly selected and their candidate target genes were analyzed by KEGG and GO.KEGG analysis showed that Upregulation of target genes involved in the gene pathway may include:P53 signaling pathway,gastric acid secretion pathway,TGF-?signaling pathway,cytokines and their receptor interactions.Target gene pathways involved in the down-regulation of genes include:chronic myeloid leukemia,proteins in cancer,signal transduction of neurotrophic factors,insulin signaling pathway,MAPK signaling pathway?AMP-dependent protein kinase?,and the like,etc..In the collected placenta sample and GEO database,the jak-stat signaling pathway was activated.Conclusion:1.The expression of mi RNA in serum and placental tissue of preeclampsia patients is significantly different from that of normal pregnant serum and placenta,indicating that there may be metabolic imbalances in the blood circulation and placenta of preeclampsia patients.2.During the pathogenesis of PE,serum miR-455-3p,miR-103a-2-5p,and miR-449c-5p are up-regulated,and miR-187-3p,miR-217,and miR-365b-5p in the placenta tissue may play an important role.3.SERPINA3 is involved in placental diseases as a protease inhibitor of elastase,which plays a crucial role during the implementation process.The activation of jak-stat signaling pathway may promote the development of PE.