| Background:Acquired immune deficiency syndrome(AIDS)is a public health problem that threatens the health of all mankind.When HIV-1 patients develop into the AIDS stage,it is very easy to merge various opportunistic infections,and Talaromyces marneffei(TM)is one of them.The incidence of TM in Guangxi AIDS patients is relatively high,and the mortality rate of those who have not been diagnosed and treated in time is very high.However,the diagnosis of TM in clinic mainly relies on pathogenic microscopy and culture,but the microscopic detection rate is low,and the fungal culture time is long,so early diagnosis for TM is difficult.Exosomes are secreted by various types of cells under physiological and pathological conditions,and contain proteins,lipids,glycans,messenger RNA and micro RNA and other components.As intercellular signaling organelles,they participate in many physiological and pathological processes,and have potential roles in clinical diagnosis and treatment.In addition,exosomes also play an important role in the physiological and pathological mechanisms during fungal infection.Therefore,this experiment intends to explore the serum exosomes of AIDS patients with TM infection,in order to find potential biomarkers for rapid diagnosis of TM and to lay the foundation for early clinical diagnosis and early treatment of AIDS patients with TM.Methods:1.According to the selection criteria,the subjects of the control group and the experimental group were included.The patient’s serum was collected and the serum exosomes were extracted by ultracentrifugation,polyethylene glycol-based precipitation method and immunomagnetic bead method,and then transmission electron microscopy and dynamic light scattering and Western blotting methods were used to identify exosomes.At the same time,the protein of exosomes was extracted and the protein concentration was determined.Finally,the protein concentration was compared between the two groups by the t test of SPSS 20.0software.2.Serum exosomes of the control group and experimental group were extracted by PEG-based precipitation method,and the RNA of exosomes was extracted by traditional Trizol method.Finally,the library was constructed by Illumina Tru Seq Small RNA kit,and SE50 was sequenced by Hiseq sequencing platform.Differentially expressed miRNAs were obtained after bioinformatics analysis.3.The miRNAs with significant differences were verified through real-time quantification polymerase chain reaction(q RT-PCR)with the additive tail method.The results were calculated by SPSS20.0 software for t test,and the variables were evaluated separately or integrated by logistics binary regression model.The receiver operating characteristic curve(ROC)was drawn to evaluate its diagnostic value.4.Target Scan Human V7.2 and star Base V2.0 databases were used to predict the target genes of the four differentially expressed miRNAs,and then the online gene function annotation tool on the DAVID website were used to analysis the differential genes for GO enrichment,in order to understand biological processes,molecular function of gene and cell component.At the same time,KEGG database was used to analyze relevant signaling pathways,and significant signaling pathways were screened with P<0.05.Results:1.In this study,35 and 33 subjects were included in the control group and the experimental group,respectively.The age,gender,white blood cell,neutrophil,lymphocyte and platelet numbers of patients were not significantly different between the control group and the experimental group,while the monocyte count,CD4~+T lymphocyte count,CD8~+T lymphocyte count and the ratio of CD4/CD8were significantly different between the two groups.The count of monocyte,CD4~+T lymphocyte,CD8~+T lymphocyte count and the ratio of CD4/CD8 in the experimental group were significantly lower than the control group,P<0.05,the difference was statistically significant.Exosomes can be successfully extracted from serum by hypervelocity centrifugation,PEG-based precipitation and immunomagnetic bead method.The particle size and distribution of exosomes extracted by the three methods showed no significant difference between transmission electron microscopy and dynamic light scattering.In addition,among the three methods,the proteins’concentration of exosome extracted by polyethylene glycol precipitation method was the highest(?X±S=62.57±4.56μg/μl,P=0.00),but the proteins’concentration of exosome extracted by the same method was no difference between the experimental group and the control group(P=0.95).2.There was no significant difference in RNA concentration of serum exosomes between the control group and the experimental group(P=0.913),and the initial template amount was the same.The error rates of the original data of exosome RNA sequencing were 0.0422%and 0.0438%,respectively.Q20 was91.38%and 90.86%in the two groups,respectively.Q30 was 82.92%and 82.23%,and the percentages of Q20 and Q30 were both greater than 80%.In addition,GC accounted for 38.17%and 38.635%of the number of bases,respectively.A total of 105768 SRNAs were obtained between the two groups.The specific SRNAs in the control group and the experimental group were 657458 and 560987respectively,accounting for 86.14%and 84.14%of the total,respectively.After comparison with RFAM database,it was found that the proportion of miRNA in non-coding RNA of the experimental group was 8%,and that of the control group was 6%.After removing non-miRNA sequences,245 known miRNAs were identified in common between the two groups,while 58 miRNAs were unique to the control group and 63 miRNAs were unique to the experimental group.By DEGSeq differential expression analysis,the miRNAs differentially expressed between the two groups were screened according to the absolute value of Log2Fc greater or equal to 1 and FDR<0.01,and a total of 73 miRNAs with up-regulated expression and 59 with down-regulated expression were screened out.3.There was no significant difference in the relative expression of the three down-regulated miRNAs(miR-144-3p,miR-199a-3p,miR-486-5p)between the control group and the experimental group,with P values of 0.294,0.459 and 0.082,respectively.P values were all greater than 0.05,the difference was not statistically significant.In addition,the relative expression level of miR-28-3p showed no significant difference between the two groups(P=0.095).The expression levels of the other four miRNAs(miR-192-5p,miR-194-5p,miR-455-3p,miR-1246)in the experimental group were significantly higher than those in the control group(P<0.05),with P values of 0.008,0.004,0.049 and 0.02,respectively.The differences were statistically significant.Further ROC curve analysis showed that the areas under the curve of miR-192-5p,miR-194-5p,miR-455-3p and miR-1246 were 0.658,0.703,0.54 and 0.721,respectively.Then the diagnostic sensitivity and specificity of the four miRNAs were calculated,and the results showed that the diagnostic sensitivity and specificity of miR-192-5p were0.649 and 0.694,respectively.The sensitivity and specificity of miR-194-5p were0.676 and 0.611,respectively.The diagnostic sensitivity and specificity of miR-1246 were 0.703 and 0.75,respectively.The diagnostic sensitivity and specificity of miR-455-3p were 0.286 and 0.966,respectively.The area under the curve of the four miRNAs combined diagnosis was 0.818,while the sensitivity and specificity of the diagnosis were 0.762 and 0.893,respectively.4.GO enrichment showed that most of the four up-regulated miRNAs were involved in the negative regulation of RNA polymerase II promoter transcription and the positive regulation of RNA polymerase II promoter transcription.In addition,positive and negative DNA transcription and cellular responses to DNA damage stimuli were also involved.The results of enrichment of molecular functions mainly involved protein binding,DNA binding,transcription factor activity and transcriptional activator activity,as well as sequence specific binding of the proximal region of the core promoter of RNA polymerase II.KEGG results showed that four miRNAs were all related to the signaling pathways regulating pluripotent stem cells,while the regulation of TGF-βsignaling pathway was significantly related to miR-192-5p,miR-194-5p and miR-455-3p.In addition,miR-192-5p was also involved in the AMPK signaling pathway;Mi R-1246 was significantly correlated with c GMP-PKG and c AMP signaling pathways.Mi R-194-5p may regulate multiple signaling pathways such as Wnt and MAPK.Mi R-455-3p may be related to AMPK and PI3K-Akt signaling pathways.Conclusion:1.In this study,serum exosomes from patients with AIDS complicated with TM infection were successfully extracted and identified,and miR-192-5p,miR-194-5p,miR-455-3p and miR-1246 with diagnostic value were screened out and verified.2.The four miRNAs may become non-invasive biomarkers of TM infection in AIDS patients,and may regulate the disease process of AIDS patients complicated with TM infection through infection-related pathways such as TGF-βsignaling pathway,AMPK signaling pathway,Wnt signaling pathway,MAPK signaling pathway,c GMP-PKG signaling pathway and c AMP signaling pathway. |
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