Role of miR-34a in HTLV-1-infected T-cells

Human T-cell leukaemia virus type 1 (HTLV-1) is the etiological agent of an aggressive neoplasm of CD4+ T-cells termed adult T-cell leukaemia/lymphoma (ATLL) and a neurodegenerative disease termed tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM). To better understand the pathways controlling HTLV-1 infection, persistence and transformation, our laboratory is investigating the interplay between the virus and the cellular microRNA (miRNA) network. This study was focused on miR-34a, which is known as a tumor suppressor in other contexts, but is highly expressed in HTLV-1 infected cell lines, newly infected PBMCs and ATLL samples. Further studies of HTLV-1-infected cell lines C91PL and MT-2 treated with the NF-κB inhibitor Bay 11-7082 indicated that the NF-κB pathway contributes to sustain miR-34a expression in this cellular context. Identification of the primary miR-34a precursor (pri-miRNA) produced in C91PL cells through the 5’RACE technique revealed that the main pri-miR-34a species present in this cell line corresponded to a two-exon transcript previously identified in cells of different lineage. The presence of an NF-κB binding site near the transcription start site of the primiR-34a provided further evidence that miR-34a expression is driven by this pathway in HTLV-1-infected cells. Treatment of C91PL and MT-2 cells with the MDM2 inhibitor Nutlin-3a resulted in stabilization of p53, a further increase in the levels of miR-34a and downregulation of several of its targets, including SIRT1, a deacetylase whose substrates include p53, the inhibitor of apoptosis protein (IAP) BIRC5, which codes for Survivin, and the transcription factor E2F3, which is involved in controlling cell cycle. Interestingly, although Nutlin-3a induced G1 arrest in both cell lines, MT-2 cells entered late apoptosis, while C91PL cells showed signs of senescence. The last part of this project was aimed at investigating the effects of Nutlin-3a on viral gene expression and directly assessing the impact of miR-34a in C91PL cells by electroporating a miR-34a mimic. We observed that Nultin-3a treatment of C91PL cells increased levels of the majority of the alternatively spliced HTLV-1 transcripts. Although the introduction of the miR-34a mimic into C91PL cells resulted in a reduction in the expression of key targets of the miRNA, it did not induce cell cycle arrest, death, senescence, or alterations in viral gene expression, suggesting that other factors come into play in the response to Nutlin-3a treatment