| Cystic echinococcosis is a worldwide chronic zoonotic disease caused by infected with the larval stage of Echinococcus granulosus.It is endemic in pastoral regions around the world.MDSCs are a heterogeneous population of myeloid cells.They accumulated in spleen and peripheral blood in the Echinococcus granulosus infected mice.MDSCs are divided into two major subsets based on their phenotypic and morphological features:polymorphonuclear(PMN)-MDSCs and monocytic(M)-MDSCs.M-MDSCs and PMN-MDSCs inhibit immune function via different mechanisms and M-MDSCs are more immunosuppressive than their counterparts when assessed on a per cell basis.While the changes and their significance of the MDSCs and the subpopulation in E.granulosus protoscoleces(Eg-psc)infected mice is unclear.In this study,we detected the percentage of MDSCs and their subpopulation in spleen,peripheral blood and peritoneal lavage by flow cytometry.And we used microarray analysis to investigate the lncRNA and mRNA expression profiles in the splenic M-MDSCs of normal and Eg-psc-infected mice,and performed bioinformatics analysis of the differentially expressed lncRNAs to explore the possible biological processes and pathways associated with M-MDSCs.The results demonstrated that aberrantly expressed IncRNAs might be new candidates behind the immunoregulation mechanism of M-MDSCs in parasitic diseases.LncRNA could regulate RNA transcripts by competing for shared miRNA response elements(MREs).This regulatory model is called competing endogenous RNA(ceRNA)and form a huge ceRNA network(ceRNETs).In this study,according to the microarray analysis of lncRNA and mRNA expression profiles in M-MDSCs in E.granulosus infected mice,combined with microarray analysis of miRNA expression profiles in M-MDSCs of the members of my group,we predicted and screened the lncRNAs that participated in the immune suppression mechanism of M-MDSCs as ceRNA in E.granulosus infected mice models by bioinformatics analysis.Part 1 Changes and significance of myeloid-derived suppressor cells in E.granulosus protoscoleces infected miceWe established E.granulosus protoscolices infected mouse model,the infected group mice were injected with 2 000 protoscoleces by peritoneal injection,while the control group mice were injected with a same volume of normal saline.Spleen cells,peripheral blood leukocytes and peritoneal cells were collected eight months after infection,in which the percentage of MDSCs and their subpopulation:M-MDSCs and PMN-MDSCs and Th17 cells were detected by flow cytometry.The correlation was determined by the Pearson correlation analysis.The results showed that the cell percentage of MDSCs of infection group in spleen cells,peripheral blood and peritoneal cells were(14.72± 4.27)%,(57.04± 6.78)%and(15.35±5.56)%,respectively,all significantly higher than those in the control group[(8.84± 2.12)%,(30.53± 1.58)%and(1.74 ±0.63)%](P<0.05).The cell percentage of M-MDSCs of infection group in spleen cells,peripheral blood and peritoneal cells were(1.29±0.24)%,(6.22±2.11)%and(2.14± 0.94)%,respectively,all significantly higher than those in the control group[(0.72±0.25)%,(2.11±1.27)%and(0.25±0.06)%](P<0.05).In the infection group,the cell percentage of PMN-MDSCs in spleen cells,peripheral blood and peritoneal cells were(9.31± 2.65)%,(46.72 ± 5.67)%and(7.06± 2.36)%,and were(7.07±3.20)%,(25.42± 2.05)%and(1.08±0.40)%in the control group.The cell percentage of PMN-MDSCs in peripheral blood leukocytes and peritoneal cells in infected group were higher than those in control group(P<0.05).The cell percentage of Th17 cells of infection group in spleen,peripheral blood and peritoneal cells were(1.31±0.38)%,(1.85±0.77)%and(2.90±0.24)%,all significantly higher than those in the control group which were(0.59± 0.07)%,(0.35±0.15)%and(0.41±0.12)%(P<0.05),Pearson correlation analysis showed that there was no correlation between the cell percentages of the Th17 and MDSCs in spleen,peripheral blood leukocytes and perntoneal cells in the infection group(r =-0.354,-0.746,0.801;P>0.05).While the cell percentage of M-MDSCs was negatively correlated with Thl7cells in spleen(r =-0.896,P<0.05).MDSCs and Th17 cells are both increased in mice at late stage of E.granulosus infection,and may play important roles in the development of echinococcosis.Part 2 Microarray analysis of long non-coding RNA expression profiles in monocytic myeloid-derived suppressor cells in E.granulosus infected miceWe compared the long non-coding RNA(lncRNA)and mRNA expression patterns between the splenic M-MDSCs of E.granulosus protoscoleces-infected mice and normal mice using microarray.LncRNA functions were predicted using Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis.Cis-and trans-regulation analysis revealed potential relationships between the IncRNAs and their target genes or related transcription factors.Results showed that 649 lncRNAs were differentially expressed(fold change?2,P<0.05):582 IncRNAs were upregulated and 67 IncRNAs were downregulated;respectively,28 upregulated mRNAs and 1043 downregulated mRNAs were differentially expressed.The microarray data was validated by quantitative reverse transcription-PCR in three other splenic M-MDSCs of normal and Eg-psc infected mice.The results indicated that mRNAs co-expressed with the IncRNAs are mainly involved in regulating the actin cytoskeleton,Salmonella infection,leishmaniasis,and the vascular endothelial growth factor signaling pathway.We found that 288 IncRNAs were considered cis-regulatory IncRNAs of their sense-overlapping genes,and 372 IncRNAs were predicted to interact with 60 transcription factors.Some IncRNAs are dysregulated in M-MDSCs;they might be involved in immunoregulation and could be potential biomarkers or novel treatment strategies for immunoregulation in related diseases.Part 3 Network analysis of competing endogenous RNA in M-MDSCs in Echinococcus granulosus infected miceLncRNA could regulate RNA transcripts by competing for shared miRNA response elements(MREs).This regulatory model is called competing endogenous RNA(ceRNA)and form a huge ceRNA network(ceRNETs).In this study,according to the microarray analysis of IncRNA and mRNA expression profiles in M-MDSCs in E.granulosus infected mice,combined with microarray analysis of miRNA expression profiles in M-MDSCs of my group members,we predicted and screened the IncRNAs that participated in the immunoregulation mechanism of M-MDSCs as ceRNA in E.granulosus infected mice models by bioinformatics analysis.And constructed the lncRNA-miRNA-mRNA regulatory network.GO analysis of the lncRNA that functioned as ceRNA,the results showed that:in the biological process,they mainly involved in the cell cycle,mitotic nuclear division,cell division;and the molecular function were involved in interleukin-1 receptor activity,catalytic activity and ATP binding;in cellular components,they mainly existed in chromosome centrometric region,chromosome and nucleus etc.KEGG pathway analysis showed that in the ceRNA regulatory network,the main signal pathways involved include:DNA replication,cell cycle,and mismatch repair.LncRNAs may regulate target genes expression as ceRNA by competitive combination of miRNA,thus affect the immunoregulation function of M-MDSCs. |
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