| ObjectiveHIV-1 infected long term non-progressors(LTNPs)could maintain stable CD4~+T cell counts over 10 years in the absence of ART.The mechanism of prolonged disease progression is considered to be associated with viral features,host genetic factors and immunological factors,however,it remains elusive.In this study,we performed an integrative analysis of transcriptional profiles between LTNPs and typical progressors(TPs)to pinpoint the underlying influence of mRNA and non-coding RNA on HIV-1 long term non-progression.Based on the differentially expressed mRNA,we further explored the role of immune activation in HIV-1 disease progression.Methods1.PBMCs were collected from 3 LTNPs and 3 TPs and subjected to RNA extraction.RNA-seq was used to obtain the expression profiles of mRNA,lncRNA,miRNA,and circRNA after cDNA construction,data filtering and annotation.Differential expression analysis was performed to identify the differentially expressed mRNAs,lncRNAs,miRNAs and circRNAs between LTNPs and TPs.2.Differentially expressed mRNAs were subjected to protein-protein interaction network analysis,GO functional enrichment analysis and KEGG pathway enrichment analysis.The target genes of differentially expressed lncRNA,miRNA and circRNA were used to perform GO functional enrichment analysis and KEGG pathway enrichment analysis.3.Based on the target genes prediction,negatively correlated miRNA-mRNA pairs and negatively correlated miRNA-lncRNA pairs were used to construct the lncRNA-miRNA-mRNA interaction network.And the circRNA-miRNA-mRNA interaction network was constructed through same processes.4.LTNPs,TPs,HIV-1 infected antiretroviral therapy-treated individuals(ARTs)and healthy controls(HCs)were recruited.PBMCs and plasma were isolated from peripheral blood samples.The mRNA expression of NLRP3、IL-1βand caspase-1 in the NOD-like receptor signaling pathway was measured by RT-PCR.The levels of pyroptosis in PBMCs between different groups were measured by flow cytometry.The plasma levels of caspase-1 and IL-1βprotein were tested by ELISA.5.The mRNA expression of cytosolic DNA-sensing pathway-associated genes was measured by RT-PCR.Western Blot assay was used to detect the protein expression of IFI16 and STING.Results1.Compared with TPs,543 differentially expressed mRNAs were identified in LTNPs,of which 205 were up-regulated and 338 were down-regulated.GO enrichment analysis showed that the differentially expressed mRNAs were involved in cellular process,cell part and catalytic activity terms.KEGG enrichment analysis showed that differentially expressed mRNAs were significant enriched in innate immunity pathways,such as cytosolic DNA-sensing pathway,NOD-like receptor signaling pathway,and TNF signaling pathway.2.A total of 1297 differentially expressed lncRNAs were identified,of which1108 were up-regulated and 189 were down-regulated.The lncRNA NRAV which negatively regulate the host antiviral immune response were down-regulated in LTNPs.3.There were 45 differentially expressed miRNAs between LTNPs and TPs,of which 8 were previously reported to have biological function.All 155differentially expressed circRNAs were novel circRNAs predicted in our study.4.The lncRNA-miRNA-mRNA interaction network included 16 lncRNAs,14 miRNAs and 64 mRNAs,harboring 100 pairs interaction relationship.The circRNA-miRNA-mRNA interaction network included 34 circRNAs,17 miRNAs and 71 mRNAs,harboring 142 pairs interaction relationship.Transcription factor TCF-7,an cirtical regulator in T-cell specification,was found in both networks.5.The mRNA expression levels of NLRP3 among LTNPs,TPs,ARTs and HC were 4.14±3.76,1.90±1.57,2.60±2.73,1.33±1.23,respectively,with a statistical significance(F=3.202,P=0.030).The mRNA expression level of NLRP3 in LTNPs was higher than that of TPs(t=2.542,P=0.015).The proportions of pyroptosis cells in four groups were all at low level,with no statistically significant difference(F=0.529,P=0.672).The caspase-1 levels in plasma among four groups had no statistically siginificant difference(F=0.852,P=0.475).In addition,there was no siginificant difference in IL-1βexpression among four groups,either at mRNA level or at protein level(P>0.05).6.There was a statistically siginificant difference in the mRNA expression of cGAS and IFI16 among four group(P<0.01).The expression of GAS and IFI16in LTNPs was significantly lower than that of TPs(P<0.01).Similarly,the mRNA expression levels of two critical factor of cytosolic DNA-sensing pathway,STING and TBK1,in LTNPs were lower than those in TPs(P<0.01).Furthermore,the protein levels of IFI16 in LTNPs and TPs were 0.19±0.08 and 0.54±0.19,which was significantly lower in LTNPs(t=2.908,P=0.044).In addition,the protein levels of STING in LTNPs and TPs were 1.85±0.73,3.37±0.47,which was significantly lower in LTNPs(t=3.015,P=0.039).Conclusion1.There are large differences in the expression profiles of mRNAs,lncRNAs,miRNAs and circRNAs between LTNPs and TPs.Bioinformatics analysis shows that innate immune pathways might involve in HIV-1 long term non-progression.Non-coding RNAs might regulate the expression of mRNAs via a ceRNA manner,and thus modulate the activation of signaling pathway.2.The NOD-like receptor signaling pathway is not activated in LTNPs.But the cytosolic DNA-sensing pathway is suppressed in LTNPs,which might be associated with prolonged disease progression... |
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