TARGETS FOR HUMAN ENCODED microRNAs IN HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE 1 (HTLV-1) tax REGION

BM15

 Nicolete, LDF (1,2) deadame@hemocentro.fmrp.usp.brAzevedo, R (2); Silva, IT (2);  Malta, TM (1,2); Zanette, DL (2); Takayanagui, OM (3);  Covas, DT (2,3); Kashima, S (1,2,3)

(1) Faculdade de Ciências Farmacêuticas de Ribeirão Preto

(2) Fundação Hemocentro de Ribeirão Preto

HTLV-1 is a retrovirus associated with two distinct diseases: HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia/lymphoma (ATLL). Only 5% of HTLV-1 infected individuals develop clinical manifestations and the risk or prognosis of diseases development is still unknown. MicroRNAs (miRNAs) are non-coding RNAs that mediate pos-transcriptional gene repression by inhibiting protein translation or by destabilizing target transcripts. Retroviruses depend on the host machinery, so they are susceptible to the host gene-regulatory mechanism. For this reason, human miRNAs (hsa-miRNAs) could be responsible for host-virus interaction and be associated with diseases. In this work, bioinformatics tools and experimental approaches have been used to find hsa-miRNAs targets in regulatory gene (tax) of HTLV-1. Firstly, we predicted hsa-miRNAs which target tax of the HTLV-1 genome using two softwares – miRanda and RNAHybrid. The algorithms were set up with personal parameters (Score 120; Mfe –20 kCal/Mol). To validate these findings, mature miRNAs expression was quantified by qRT-PCR employing stem-loop primers strategy. For this purpose, total RNA from PBMC cells was obtained from HTLV-1 patients classified according to the clinical status: asymptomatic (HAC) (n=6) and HAM/TSP patients (n=9). Proviral load (CPV) was measured for all HTLV-1 positive samples. A control group (negative samples, n=6) was also included. Bioinformatics analysis demonstrated that several miRNAs presented good results for established parameters, but hsa-mir-221 (Score, 159 Mfe –21,4 kCal/Mol); hsa-miR222 (Score 168, Mfe –30,5kCal/Mol) and hsa-miR-125b (Score 138, Mfe –25,6kCal/Mol) were used for initial investigation. For validation approaches, control group was used as calibrator. We obtained high levels of hsa-mir- 222 expressions in HAM/TSP group (more than 2.5 times) in contrast to the other groups. Hsa-miR-125b presented higher levels in patients than controls (more than 10 times). Additionally, statistical analysis showed a correlation between this hsa-miR-125b and CPV (p=0,0428). Until now, these miRNAs (hsa-mir-221, hsa-miR222 and hsa-miR-125b) were associated to cancer. This is the first study attempting to the differential expression of miRNAs in HTLV-1 related diseases. (FAPESP -06/59388-4), (CNPq -475091/2006-0), (CTC/FUNDHERP).