Large-scale screening of circulating microRNAs in individuals with HIV-1 mono-infections reveals specific liver damage signatures

Human immunodeficiency virus type 1 (HIV-1)-induced inflammation and/or long-term antiretroviral drug toxicity may contribute to the evolution of liver disease. We investigated circulating plasma microRNAs (miRNAs) as potential biomarkers of liver injury in patients mono-infected with HIV-1. We performed large-scale deep sequencing analyses of small RNA level on plasma samples from patients with HIV-1 mono-infection that had elevated or normal levels of alanine aminotransferase (ALT) or focal nodular hyperplasia (FNH). Hepatitis C virus (HCV) mono-infected patients were also studied. Compared to healthy donors, patients with HIV-1 or HCV mono-infections showed significantly altered (fold change >2, adjusted p<0.05) level of 25 and 70 miRNAs, respectively. Of the 25 altered miRNAs found in patients with HIV-1, 19 were also found in patients mono-infected with HCV. Moreover, 13 of the 14 most up-regulated miRNAs (range: 9.3-3.4-fold increase) in patients with HCV mono-infections were also up-regulated in patients with HIV-1 mono-infections. Importantly, most of these miRNAs significantly and positively correlated with ALT and aspartate aminotransferase (AST) levels, and liver fibrosis stage (p<0.05). MiR-122-3p and miR-193b-5p were highly up-regulated HIV-1 mono-infected patients with elevated ALT or FNH, but not in HIV-1 patients with normal levels of ALT. These results reveal that HIV-1 infections impacted liver-related miRNA levels in the absence of an HCV co-infection, which highlights the potential of miRNAs as biomarkers for the progression of liver injury in HIV-1 infected patients. Overall design: In total 97 plasma derived small RNA samples were analyzed: 21 from healthy reference and 76 from virally mono-infected individuals. There were two patient groups based on virus infection status: a group of 22 individuals infected with hepatitis C virus (HCV), and a group of 54 individuals infected with human immunodefficency virus (HIV). The study focused on determining miRNA expression differences in the context of liver damage. Among HIV mono-infected there were 22 with normal ALT levels, 21 with ALT levels above the upper limit of normal and 11 with biopsy proven nodular regenerative hyperplasia (NH). All samples were controlled for hemolysis using a qPCR based test taking only samples with miR-451/miR-23a ratios with ?Ct<7 (range -0.04 to 6.3; average 3.53). Design efforts were made to randomize all subgroups to be equally represented in the library preparation and size selection steps to be able to correct for possible batch effects. The experiment was performed in the context of a larger sample set including a total of 144 samples. Size selection was performed in pools of 24 libraries, and 3 sets of 2 library pools were merged into 3 superpools of 48 samples each for Illumina sequencing. In the final analysis 6 samples were excluded from the final differential miRNA expression analysis as they showed outlier behavior in unsupervised exploratory pricipal components analysis: 5 HCV monoinfected and 1 HIV infected with elevated ALT. Thus, final results of this work were derived from 91 samples comparing: 21 healthy, 17 HCV monoinfected, 22 HIV mono-infected with normal ALT, 20 HIV mono-infected with elevated ALT, and 11 HIV mono-infected with NH.