Análisis de los perfiles de expresión de miARN y caracterización funcional en individuos VIH-positivos con distinta progresión a SIDA

Laburpena

About 1% of people living with HIV (PLWH) are elite controllers (EC), competent to maintain their viral load below 50 copies of viral RNA/ml for long periods of time in the absence of antiretroviral therapy (ART). On the other hand, long-term non-progressors (LTNP) are able to maintain CD4+ T-cell numbers above 500 cells/µL for more than 10 years without ART and represent less than 5% of PLWH. Both phenotypes in combination (EC-LTNP) represent an excellent natural model for functional cure of HIV infection. Although several factors involved in long-term non-progression and natural control have been described, the role of miRNA has not been investigated in depth. The first objective of this work is to study the miRNome of PLWH with extreme phenotypes and to identify biological processes potentially altered by differentially expressed (DE) miRNA. The miRNome was obtained by next-gen sequencing (miRNA-Seq) of peripheral blood mononuclear cells (PBMC) isolated from EC-LTNP, viremic LTNP (<10,000 cp/ml) and PLWH with a typical progression profile (TP). Upon analysis, repression of miR-144-3p, miR-486-3p and miR-3607-5p has been associated with the EC-LTNP phenotype compared to TP with suppressed viral load under ART. In addition, 14 miRNA differentiate LTNP from TP. Four of these miRNA (miR-144-3p, miR-18a-5p, miR-451a, and miR-324) are strongly downregulated in LTNP and regulate the expression of AKT, mTOR, ERK, and IκK, genes involved in the immune response. Subsequently, the second objective of this Thesis is to identify a molecular signature based on the miRNome and transcriptome of CD4+ T cells from EC. In addition, the regulatory role of DE miRNAs on gene expression and the evaluation of miRNA antiviral activity by functional assays are studied. Transcriptome and miRNome, in addition to other non-coding small RNAs (sncRNA), were obtained by RNA-Seq of CD4+ T cells from EC, viremic controllers (<4,000 cp/ml), and non-controllers off (NT) and on ART (ART). The miRNA, mRNA and other sncRNA associated with the natural control of HIV infection, as well as the main pathways and biological terms altered in this phenotype have been studied by differential expression analysis and modeling with Projections on Latent Structures (PLS) using Gene Set Enrichment Analysis (GSEA) and Overrepresentation Analysis of DE Genes (ORA). The miR-99b-5p and miR-125a-5p were downregulated in EC compared to the ART group. In contrast, comparing EC versus NTs, overexpression of miR-27a-5p has been described along with the attenuation in the expression of miRNAs, including miR-99b-5p and miR-125a-5p. We searched for the DE miRNA gene targets with reverse differential expression and constructed regulatory networks, revealing that targets of miR99b-5p (TIMP2, MAFB, MMP9, DUSP1, or CCL3) and miR-125a-5p (LAMP1, CDKN1A, NBEAL2, BBC3, CSRNP1, SOD2) participate in p53 signaling pathways, the phagosome machinery, or reactive oxygen species production. In addition, the transcriptome of CD4+ T cells from EC shows increased cellular activation and inflammatory response, as well as increased oxidative stress buffering capacity and decreased expression of genes containing KRAB and C2H2 motifs associated with Zinc fingers. Treatment with miR-99b and miR-125a-5p inhibitors in vitro decreased HIV-1 viral production. The results presented in this Thesis indicate that miRNA have an antiviral role in the natural control of HIV infection and long-term progression to AIDS and although they are not on their own fully responsibles for the development of these phenotypes, they are key players in the modulation of the transcriptional landscape of cells, helping to the maintenance of the CD4+ T cell count and hindering virus replication. The study of their gene targets and the metabolic and signaling pathways affected by them may provide new therapeutic and monitoring opportunities for their use in the clinical setting.