Analysis Of Transcriptome Gene Expression Profile And Screening Of Hub Genes In Unexplained Recurrent Spontaneous Abortion

[Objective]The transcriptome gene expression profile of decidual tissue of unexplained recurrent spontaneous abortion（URSA）was analyzed by RNA-seq technique to find the potential key pathogenic genes of URSA.Using SNP Microarray to screen the Polymorphisms of Gene loci affecting the occurrence of URSA.Flow cytometry was used to detect the difference in the proportion of Treg cells and three Treg cell subsets in patients with URSA.This lays an experimental foundation for the study of immune mechanism of URSA,provides new ideas and targets for its clinical diagnosis and treatment.[Method]1.Sample collection.A total of 46 pregnant women who were admitted to Changsha Maternal and Child Health Hospital from October2018 to December 2020 were collected,including 23 cases in URSA group and 23 cases in normal pregnancy control group.Samples of decidual tissue and peripheral blood were obtained for follow-up experiment.2.Transcriptome gene sequencing.The total RNA of decidual tissue of 8 pregnant women with URSA and 4 pregnant women with normal pregnancy were sequenced with high throughput.3.Bioinformatics analysis.The transcriptome gene expression profiles of the two groups were compared and analyzed,and the differentially expressed genes of URSA were screened.The function and characteristics of differentially expressed genes were analyzed by GO and KEGG enrichment analysis.The PPI network of differentially expressed genes was constructed,and the key hub genes were screened by analyzing the interaction between genes.All genes in URSA group and normal control group were analyzed by GSEA,and the gene sets closely related to phenotype were found.4.GSE113790 dataset analysis.Search the GEO database to obtain the transcriptome sequencing data set related to URSA GSE113790,for bioinformatics analysis.The results were compared with the self-sequencing data to prove whether the selected key genes and pathways were accurate.Combined with the RNA-seq sequencing data of the two groups,WGCNA analysis was carried out to find out the gene modules most related to the phenotype of URSA.5.RT-PCR test.Total RNA was extracted from decidual tissues of 23cases of URSA and 23 cases of normal pregnancy control.Four key hub genes with statistically significant differences were detected by RT-PCR to confirm whether their expression trend was consistent with the results of transcriptional sequencing.6.SNP chip detection.ASA-CHIA whole genome SNP microarray was used to detect the peripheral blood samples of 6 URSA groups and 9normal pregnant controls.The single nucleotide polymorphisms of key genes were analyzed,and the allele frequencies between the two groups were compared.7.Detected by flow cytometry.Flow cytometry was used to detect the levels of Treg cells and three Treg cell subsets（r Treg,a Treg and cs Treg）in the peripheral blood of URSA group（n=23）and normal pregnancy group（n=23）.[Results]1.There was no significant difference in the general clinical data between the URSA group and the normal pregnancy control group（P>0.05）,indicating that the sample selection met the admission criteria.2.The results of bioinformatics analysis showed that a total of 1747differentially expressed genes（DEGs）,were screened using|log2 Fold Change|>2,P value<0.01.among them,958 were up-regulated and 789were down-regulated.GO enrichment analysis showed that the main biological processes of DEGs enrichment included:leukocyte chemotaxis,T cell activation and differentiation,negative regulation of immune system response,reproductive system development（P<0.05）;cell components items:MHC protein complex and so on（P<0.05）;molecular function items:receptor ligand binding activity,transcription factor activity and so on（P<0.05）.KEGG and GSEA enrichment analysis showed that the main signal pathways of significant enrichment were complement and coagulation cascade reaction,antigen processing and presentation,autoimmune disease,FOXP3 protein activation pathway,CTLA4 signal pathway,PI3K-AKT signal pathway,IL10-anti-inflammatory signal pathway,JAK2 signal pathway and so on（P<0.05）.These genes and pathways are mainly related to the immune status of the body and the proliferation and function of Treg cells.Nine key central genes such as CXCL9,CXCL10,GPR183,XCL1,EGR1,HLA-DRB1,EPHA2,CCL8and WASL were screened by PPI network analysis and WGCNA analysis.3.Bioinformatics analysis of GSE113790 data sets showed that,compared with the results of sequencing data analysis in this study,the key genes and signal pathways mainly enriched between the two were basically the same.4.The results of RT-PCR detection showed that the expression level and trend of four differentially expressed genes such as GPR183,XCL1,HLA-DRB1 and EGR1 were consistent with the results of transcriptome gene sequencing.5.The results of SNP-array detection showed that the allele frequencies of 14 key nodes,such as NOTCH3,HLA-DPA1,TNFSF8,IL2RA,SLC15A1,SPRN,PDE6H,UST,SBK1,CCDC85A,COLEC12,TMEM65,BMP6 and FAM110B,were significantly different between the two groups（P<0.05）,which may be related to the risk of URSA.6.The results of flow cytometry showed that the levels of Treg cells and CD25^hiCD45RA^-activated Treg cells（a Treg）in peripheral blood of URSA patients were not significantly different from those of the control group（P>0.05）,but the levels of CD25⁺CD45RA⁺resting Treg cells（r Treg）which played an potential immunosuppressive role were significantly lower than those of the control group（P<0.05）.,while the levels of CD25⁺CD45RA^-Treg cells（cs Treg）secreting inflammatory cytokines were significantly higher than those of the normal control group（P<0.05）.[Conclusion]1.A total of 1747 differentially expressed genes of URSA were screened by transcriptome gene expression profile,of which 9 key genes（CXCL9,CXCL10,GPR183,XCL1,EGR1,HLA-DRB1,EPHA2, CCL8,WASL）were significantly down-regulated in URSA patients,and are closely related to the activation of Treg cells and immunosuppressive function.2.SNP gene chip detection showed that 14 genes NOTCH3,HLA-DPA1,TNFSF8,IL2RA,SLC15A1,SPRN,PDE6H,UST,SBK1,CCDC85A,COLEC12,TMEM65,BMP6 and FAM110B had locus polymorphism in URSA group.3.The proportion of cs Treg cells secreting inflammatory cytokines in peripheral blood of patients with URSA is significantly increased,while the proportion of r Treg cells that can play an potential immunosuppressive role is significantly decreased.The change of the proportion of these Treg subsets may be closely related to the occurrence of URSA.