High-throughput Sequencing Combined With Bioinformatics Analysis Of MiRNA Expression Profiles In Peripheral Serum Of Patients With Severe Preeclampsia And Screening Of Possible Markers

Objective:To analyse miRNA expression profiles in peripheral circulating serum of patients with severe preeclampsia(SPE)and screen key miRNAs in differentially expressed miRNAs,via high-throughput sequencing technology and bioinformatics methods.To analyse the target genes of the key miRNAs and related molecular mechanisms in SPE and maternal-fetal-related complications and try to explore their potential as disease markers initially.Methods:1.Five patients with SPE and 5 healthy pregnant women in the third trimester were included.The peripheral venous blood was centrifuged to obtain serum.Mi RNAs were extracted using kits and sent to a sequencing company.After passing the quality inspection,a c DNA library was constructed for high-pass Quantitative sequencing and analysis of sequencing results.2.Sort the top 5 miRNAs with the largest differences according to the P value,search for target genes in the miRNA integration database mir DIP,and compare the search results with the SPE patients in the high-throughput gene expression database(GEO).The differential genes in the placental tissue gene expression data set were subjected to cross analysis.The genes in the cross set were target genes that circulating miRNAs may directly regulate and affect placental function.The function and pathway enrichment analysis of these target genes was carried out,and those closely related to placental function were selected as candidates for key genes,miRNAs that regulate these candidate key genes were identified as candidate key miRNAs for further analysis.3.Using bioinformatic methods to analyze the three PE-related datasets in the GEO database,a ce RNA network of circ RNA-miRNA-m RNA in placental tissue was constructed.The expression levels in normal human tissues and related function information of these candidate genes was searched in the Gene database of the National center for biotechnology information(NCBI),to further screen key genes,and the related miRNAs were identified as key miRNAs.Then discuss the functions of these key genes in combination with literature,analyze the key miRNAs in circulation directly affect placental function by regulating key genes.The molecular mechanisms that may be involved in the occurrence and development of SPE and related maternal and neonatal complications,and its potential as a relatively non-invasive diagnostic marker for SPE or maternal and neonatal related complications were initially explored.Results:1.The content and quality of free total RNA in the submitted serum samples meet the sequencing requirements and qualified for high-throughput sequencing;the small RNAs obtained by sequencing include miRNAs with known sequences and miRNAs that have not been compared to the database but can be determined according to the precursors.The new miRNAs predicted by the hairpin structure;with p value<0.05 and |log2FC|>1 as the screening threshold,a total of 83 known miRNAs were differentially expressed between the two serum groups,of which 42 were highly expressed and 41 were low expressed.2.The top 5 miRNAs with the greatest differences were hsa-miR-1303(low expression),hsa-miR-3122(high expression),hsa-miR-3150a-5p(low expression),hsa-miR-3157-3p(low expression)and hsa-miR-627-3p(low expression),a total of 750 predicted target genes.Cross-analysis of predicted target genes with differential genes in dataset GSE147776 resulted in 4 genes: ESRRG and AFF1(regulated by hsa-miR-3122),EGLN3(regulated by hsa-miR-3150a-5p)and PTPN22(regulated by hsa-miR-3122)3157-3p regulation),as possible key target genes and candidates for key miRNAs in SPE.3.Based on the datasets GSE147776,GSE96984,and GSE103542,we successfully constructed a ce RNA regulatory network of circ RNA-miRNA-m RNA in placental tissue.The results showed that the two target genes ESRRG and AFF1 regulated by the ce RNA network in SPE were both low-expressed,which was different from previous ones.The analysis results in peripheral blood were consistent.Combined with the Gene database search results,ESRRG and AFF1 were both highly expressed in normal placental tissue.Therefore,it is believed that ESRRG and AFF1 may be closely related to the occurrence and development of SPE,while regulating the hsa-miR-3122 may be a key miRNA in SPE.4.It is reported that ESRRG is related to PE and IUGR by regulating placental function.AFF1 is highly expressed in normal placenta,and its role in PE remains to be studied.As a miRNA that regulates ESRRG and AFF1,hsa-miR-3122 is differentially expressed in circulation,especially in peripheral blood,making it a potential molecular marker for SPE diagnosis and assessment,which deserves further study.Conclusions:1.There are stable free miRNAs in the serum of patients with severe PE,and their levels are different from those of normal pregnant women.2.Among the differential miRNAs,hsa-miR-3122,hsa-miR-3150a-5p and hsa-miR-3157-3p were candidate miRNAs for key miRNAs in SPE.3.ESRRG is closely related to SPE and IUGR by affecting placental function.AFF1 is highly expressed in normal placenta but lowly expressed in SPE placenta,and its role in PE remains to be studied.Hsa-miR-3122,which simultaneously regulates both genes,may be a key miRNA in SPE.4.hsa-miR-3122 has the potential as a relatively non-invasive molecular marker for diagnosis and assessment of SPE.