| ObjectiveTo screen and verify plasmic microRNA?miRNA?as biomarkers which can distinguish HIV-1 new infections from old infections,and establish a new detecting technique to identify HIV-1 new infections.MethodsStudy subjects were recruited from Centers for Disease Control and prevention?CDCs?of Nanning,Liuzhou,Guigang,Qinzhou,Congzuo inGuangxi,and Guangxi Medical University,between August 2014 and December2015.Participants were divided into screening set and verification set based on recruited time,and each set including health controls,HIV-1 new infections and HIV-1 old infections.Participants were verbally informed of the purpose andcontent of the study.All participants were enrolled after getting their signed informed consents.Information of estimated duration of infection?EDI?,transmitted routes,et al.,and peripheral venous blood were collected.Differential expression of plasmic miRNA of subjects in screening set were screened by microarray and further verified in verification set by real-timeq-PCR.HIV-1 IIIB-MT 2 infection system were established,and MT2 cells and supernatant were collected at day 1,day 2,day 4 and day 8.miRNAs extracted from MT2 cells and supernatant were used to verify differential expression in vitro.Comparison of miRNA expression among health controls,HIV-1 newinfections and HIV-1 old infections were performed by one-way ANOVA.Correlation between levels of miRNAs from MT2 cells and supernatant and infection time were tested by Pearson correlation analysis.Receiver operating characteristic?ROC?curve analysis of miRNA were performed by MedCalc.Results1.Fifty-one subjects were enrolled in this study,most of them were male.There were thirty participants in screening set?ten health controls,ten HIV-1new infections and ten HIV-1 old infections?and twenty-one in verification set?seven participants in each group?.The percentage of male in all participants was 98.04%.Distribution of gender in each group in both sets was notsignificantly different.HIV-1 infected subjects were mainly transmitted byhomosexual behaviors?44.12%?and by heterosexual behaviors?35.29%?.Most HIV-1 new infections were transmitted by homosexual behaviors?88.24%?.While,most HIV-1 old infections were transmitted by heterosexual behaviors?58.82%?.2.The average EDIs of HIV-1 new infections in screen set was 180±100days,the average EDI of HIV-1 old infections were 1228±531 days.Theaverage EDIs of HIV-1 new infections and old infections were 175±116 days and 1431±904 days in verification set,respectively.In these two sets,thedifference of EDI of HIV-1 new infections had no statistical significance.And the difference of EDI of HIV-1 old infections between these two sets was not significant,too.However,in both sets,the EDIs between HIV-1 new infections and HIV-1 old infections were significantly different.3.Differential expression of miRNAs was detected by Microarraytechnique.299 mi RNAs were differentially expressed between HIV-1 new infection and health controls,107 miRNAs were differentially expressed between HIV-1 old infections and health controls,and 299 miRNAs weredifferentially expressed between HIV-1 old infections and HIV-1 new infections.Based on the differentially expressed miRNAs between HIV-1 old infection and HIV-1 new infection,19 human miRNAs were filtered to be expressed withcertain trends among health controls,HIV-1 new infections and HIV-1 oldinfections.Of the 19 miRNAs,5 were differentially expressed with up-trends,14 were expressed with down-trends.4.Another 21 participants were recruited as verification set.Real-time quantitative PCR was applied to verify the differential expression of the 19miRNAs.The results of verification indicated that miR-3609,miR-148a-5p and miR-1291,which were significantly up-regulated in screening set,showeddown-trends on the order of health controls,HIV-1 new infections and HIV-1old infections.Only 2 of the 14 down-regulated miRNAs in screening were detectable in verification set.Luckily,miR-874-5p and miR-3162-3p showed down-trends in both screening set and verification set.However,the differential expressions of miR-874-5p and miR-3162-3p among health controls,HIV-1new infections and HIV-1 old infections were not significant.5.In the MT2 cells of HIV-1 IIIB-MT 2 infection system,only mi R-1291and miR-3609 were detected.However,in the supernatant of HIV-1 IIIB-MT 2infection system,only miR-3609 and miR-3162-3p were detectable.Theexpression of miR-3609 in MT2 cells of HIV-1 IIIB-MT 2 infection system was positively correlated with HIV infected time?r=0.984,95%CI:0.4265,0.9997,P=0.0158?.Moreover,the expression of mi R-3162-3p in the supernatant ofHIV-1 IIIB-MT 2 infection system was negatively correlated with HIV infected time?r=-0.9984,95%CI:-1,-0.921,P=0.0016?.6.Result of ROC curve analysis indicated that the best cut-off value of miR-3162-3p level(2-?Ct)was 0.916,at which miR-3162-3p could distinguish HIV-1 new infections from HIV-1 old infection.Sensitivity and specificity for miR-3162-3p to distinguish new infection from old infection were 100.0%and71.43%,respectively.ConclusionIn this study,differentially expressed miRNAs were screened byMicroarray technique,and were further verified in verification set and HIV-1IIIB-MT2 infection system.The findings of this study showed thatmiR-3162-3p in plasma not only exhibits differential expression among healthy controls,HIV new infections and HIV old infection,but also demonstrated anegative correlation with the duration of HIV infection.miR-3162-3p in plasma could be used as the potential biomarker of HIV-1 new infection. |
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