The role of microRNAs in HTLV-1 infection

Author(s)

Larissa Deadame de Figueiredo Nicolete

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2009-02-27

Examining board members

Simone Kashima Haddad; Victor Hugo Aquino Quintana; Osvaldo Massaiti Takayanagui

• Advisor: Simone Kashima Haddad

Abstract

MicroRNAs (miRNAs) are simple strand RNA (22 nucleotides), which are regulators of cellular gene expression. Recent studies have been showed miRNAs role as modulators of cellular responses, when viral infection occurs. The human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that presents the same regular structural genes compared to the other retrovirus, besides tax and rex genes, which encode regulatory proteins. In regard to this, Tax oncoprotein is a major determinant of viral persistence and pathogenesis. Two major diseases are related to this virus adult T cell leukemia (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Host-related factors are also correlated with HAM/TSP development. To establish if human miRNAs could influence in the unregulated cellular process caused by Tax (differentiation, signal transduction, apoptosis, cellular proliferation and tumorigenesis), this study analyzed miRNA expression in peripheral blood mononuclear cells (PBMCs) from HTLV-1 infected patients. For this purpose, we performed in silico analyses to find human miRNAs that could targeted HTLV-1 tax region. We performed two algorithms (miRanda e RNAhybrid) and we set up two distinct parameters: score more than 120 in the seed region and minimum free energy until -15 kcal/mol. So, we have selected these hsa-miRNAs:149a, 648, 221, 222, 142-5p, 26a, 29a, 374 e 125b. Besides this, we compared several HTLV-1 sub-types and we observed that regions, where miRNAs are regulating, are extremely conserved. After the in silico analysis, we validated miRNAs that we have been chosen. We isolated PBMC samples from 21 control-individuals (CT), 10 from asymptomatic carriers and 12 from HAM/TSP (HAM). After the procedure, genomic DNA were extracted from 1x106 cells, in order to quantify the proviral load (PL) performing quantitative Real-Time PCR (qRT-PCR). Tax was measured by flow cytometry and for this procedure, we used 1x106 criopreserved cells. Amount 1x 107cells were used to total RNA extraction that was used to miRNAs validation by qRT-PCR. The organic phase from Trizol® was employed to extract total proteins, which were used to analyze Tax expression by Western blot. The PL was significantly higher in HAM group than HAC (p= 0,0046), when Mann-Whitney statistical test was employed. The Tax expression was significantly correlated to PL (p=0,0128) by Spearmann test and this finding provided us a better characterization of the patients. The miRNAs expression was detected in the following groups: CT vs Patients and CT vs HAC vs HAM by non-parametrical tests Mann-Whitney and Kruskal-wallis, respectively. The miRNAs presented unregulated themselves in the patient group, when we compared to CT group. We observed that hsa-miR-125b expression are significantly higher in patients group than controls (p=0,0285). We have tried to associate miRNAs with Tax expression, employing Spearmann test, but this analysis did not show significant values. Despite this, hsa-miR-125b is responsible for regulating several proteins, such as TNF-, which is an inflammatory protein overexpressed in HTLV-1 carriers. In conclusion, we propose that this miRNA could be increased as a way to inhibit TNF- translation. These results suggest the importance in understanding how human miRNAs could regulated the HTLV-1 infection. In addition to this, they could generate data to be used as therapeutic strategies for the control of viral replication and pathogenic processes. (AU)