Maternal Placental MiRNA Profile Analysis And Application In Early-onset Severe Preeclampsia With Intrauterine Growth Restriction Based On High-throughput Sequencing

ObjectivesThe expression of miRNAs in placental tissues of patients and normal pregnant women,using bioinformatics analysis methods,compared miRNA different expression profiles,to predict target gene of miRNAs with significant different expression by Targetscan and mi DRB,GO enrichment analysis and KEGG Pathway enrichment analysis of target genes involved in signaling pathways and biological significance,determined the biological function of differently expressed miRNAs in the pathogenesis of PE,and predicted potential new miRNAs.RT-PCR was used to verify the target miRNAs,and the HIF-1?signal pathway with the highest enrichment score and the PI3K-Akt signaling pathway which involves the largest number of target genes,were analyzed to explore the possible mechanism of its effect on the development of s PE combined with FGR and fetal prognosis.methods1.Samples were collected from a total of 10 pregnant women who deliver single fetus by cesarean section in the Department of Obstetrics and Gynecology,including 5 patients with early-onset s PE and FGR,and 5 normal pregnant women.2.Total RNA was extracted from the collected placental tissues using high-throughput sequencing technology,and adapter sequences were ligated at the 5?and3?ends of miRNAs using ligase,respectively,for RT-PCR amplification,from which small RNAs of 15-35 nt were isolated and purified,and the library was denatured into a c DNA library.Sequencing was then performed with the Illumina method,and the corrected data(trimmed data)were collected by processing the data through sequencing data quality control(QC),preprocessing,etc.3.The resulting trimmed data were compared with the miRBase database,so as to obtain information on known miRNAs in the database,including:length,structure,and expression level of miRNAs.4.The expression differences of miRNAs were compared,and differential expression analysis of miRNAs was performed using edge R software to calculate P<0.05 and the CPM?1,and fold change>1.5 was selected to screen differently expressed miRNAs.5.Through bioinformatics analysis,to study the target genes of differently expressed miRNAs,GO enrichment analysis and KEGG pathway analysis of the target genes,and predicting new m RNAs by miRDeep2.6.The Real time fluorescent quantitative polymerase chain reaction(RT-PCR)was performed to examine target miRNAs.Six up-regulated target miRNAs(miR-105-5P,miR-196a-5p,miR-31-5p,miR-210-5p,miR-498-5p,miR-10b-5p)and one down-regulated target miR-200a-3p were selected for verification.The relative expression levels of miRNAs in different groups were analyzed by 2-^??Ct.7.Deep analysis of the highest enrichment of HIF-1?signaling pathway and the PI3K-Akt signaling pathway which involves the largest number of target genes,focusing on the key miRNAs and related proteins involved in these pathway,to explore its association with early-onset s PE combined with FGR.Results1.A total of 841 known,mature human miRNAs were detected,and a total of52 miRNAs(P<0.05)were significantly up-regulated and 69(P<0.05)were significantly down-regulated in placental tissues of s PE pregnant women.2.To identify and analyze differently expressed miRNA target gene,and based on the target gene database,GO functional enrichment analysis and KEGG enrichment analysis were performed on the target genes regulated by miRNAs,and these miRNAs were widely involved in the post-transcriptional regulation of gene expression,and KEGG enrichment analysis showed that the target gene regulatory pathways involved in up-regulated miRNAs include:HIF-1?signaling pathway,RNA transport,cell junction structure,insulin signaling pathway and so on.The target gene pathways involved in down-regulating miRNAs include:ascorbic acid and aldose metabolism,pentose and glucuronide interconversion,bacterial invasion of epithelial cells,PI3K-AKt signaling pathway,porphyrin and chlorophyll metabolism and so on.3.Potential new m RNAs were predicted by miRDeep2,and the location of new miRNAs on the genome,as well as the corresponding nucleotide sequence,was found by simple processing of miRNAs by Dicer to predict new miRNAs with high confidence.4.miRNAs(miR-105-5p,miR-196a-5p,miR-31-5p,miR-210-5p,miR-498-5p,miR-10b-5p,miR-200a-3p)were validated using RT-PCR,suggesting that their expression results were basically consistent with those of high-throughput sequencing,There were significant differences in miR-105-5p?miR-196a-5p and so on(P>0.05)?Conclusion1.The expression of miRNAs in Placenta of early-onset s PE with FGR was significantly different from that of normal pregnant women.RT-PCR also confirmed that the trend of miRNA in high-throughput sequencing was consistent with the actual expression of miRNA in Placenta,it is suggested that miRNA may be involved in the development of s PE disease and affect fetal prognosis.2.miRNAs are widely involved in the regulation of gene expression,and the target gene regulate pathways and predicted potential new miRNAs can provide new theories and targets for the study of Pathogenesis.3.The response mechanism mediated by the HIF-1?signal pathway which has the highest enrichment score is closely related to hypoxia stress and vascular disorder,the PI3K-Akt signaling pathway,which involves the largest number of target genes,contains 54 genes and may simulate the key process of s PE multi-link and multi-pathway pathogenesis.And miR-196a-5p positively regulates the HIF-1 signal pathway through the target gene EGLN1,which demonstrate the feasibility of regulating the abnormal expression of miRNAs in s PE pathogenesis once again.