Comparison of methods and characterization of small RNAs from plasma extracellular vesicles of HIV/HCV coinfected patients

Abstract

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) hijack the host exosomal machinery as an additional mechanism of infection and evasion of the immune system, modifying the small RNA (smRnA) cargo during infection. We characterized the surface epitopes of extracellular vesicles (eVs) from plasma HIV/HCV-coinfected patients and their smRNA cargo profile, by comparing different isolation procedures. Six EVs isolation procedures were compared: ultracentrifugation, and five different polyethylene glycol-based methods (commercial, combined with a column purification step and two custom); and two RNA commercial kits (phenol and non-phenol based) were used. Highthroughput sequencing of smRNAs was performed. Exosomal surface epitopes were analyzed by the MACSPlex Exosome Kit. Four miRNAs displayed differences among protocols (hsa-miR-205-5p and hsa-let-7a/b/f-5p). The selection of RNA isolation kit impacted on the detection of miRNAs and other smRNAs, where the phenol-based RNA isolation kit performed acceptably. EVs surface was enriched with HLA-DR/DP/DQ, CD81, and CD8. There were three liver-specific miRNAs overexpressed (let-7a-5p, miR-21-5p and hsa-miR-122-5p), thus, EVs cargo might reflect liver disease evolution. Other smRNAs such as piwi-interacting RNAs were also detected for the first time. Custom polyethylene glycol precipitation-based methods combined with an RNA phenol-based kit yielded the higher number of smRnAs for eVs isolated from plasma HiV/HcV patients.

Extracellular vesicles isolated by size-exclusion chromatography present suitability for RNomics analysis in plasma

“…This is the most systematic work that comparing EVs isolation methods in the respect of RNomics. Other researches on the comparison of RNA extracted from EVs, were that comparison of miRNA and mRNA from urinary extracellular vesicle extracted by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC), and a commercial kit‐based isolation method (NG) [ 37 ], or comparison of miRNA from plasma samples EVs of healthy dogs by ultracentrifugation, precipitation, and membrane affinity chromatography methods [ 38 ], or comparison of miRNA from plasma extracellular vesicles of HIV/HCV coinfected patients by ultracentrifugation and precipitation methods [ 39 ], or comparison of total RNA and mRNA from Whole Blood EVs extracted by only two methods including ultracentrifugation (UC) and Exodisc, a fully integrated centrifugal microfluidic device, which has previously been demonstrated to enrich EVs from urine and cell-culture supernatant [ 40 ]. In addition, few studies report the comparison of extraction exosomal lncRNA by these four different methods SEC, exoEasy, UC and ExoQuick.…”

Interference between miR-21/PTEN/E-Cadherin and Epithelial-Mesenchymal Transition in Various Stages of Chronic HCV Infection

“…Our data agree with the previously reported study of Lendvani et al [48], who proved that the expression level of miR-21 is significantly elevated in HCC patients. miR-21 is also overexpressed in hepatic tissues from HCV-infected patients [49][50][51][52] ] and hepatic cancer cell lines [53] . Our data agreed with Wang et al [54] data on the same track.…”

The involvement of microRNAs in HCV and HIV infection

“…hsa-miR-205-5p and hsa-let-7a/b/f-5p, but their role in HIV-HCV co-infection has not been studied yet. 105 Similarly miR-149 is seen to have noticeable upregulation during HCV infection. 106 At the same time, it is also observed to anneal with HIV 3′ UTR and protein Vpr during HIV infection.…”